Molecular Cell Biology (Lodish, Sixth Edition)

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About the cover: Mitotic PtK2 cellsin late anaphasestainedblue for DNA and greenfor tubulin. Courtesyof Torsten\gittman. Library of CongressCataloging-in-PublicationData Molecular cell biology I Harvey Lodish . . . [et al.]. -6th ed. p. cm. Includesbibliographicalreferencesand index. 1. Cytology. 2. Molecular biology. I. Lodish, Harvey F. QH581.2.M6552007 57L5-dc22 2007006188 ISBN-13: 97 8-0-7167-7601-7 ISBN-10:0-7167-7 601-4 @ 1986,1.990,1995,2000,2004,2008 by tJf.H. Freemanand Company All rights reserved. Printed in the United Statesof America Secondprinting W. H. Freemanand Company 41 Madison Avenue,New York, NY 10010 Houndmills, Basingstoke RG21 6XS, England

www.whfreeman.com

To our studentsand to our teachers, from whom we continueto learn, and to our families,for their support, and love encouragement,

PREFACE

I n writing the sixth edition of Molecular Cell Biology I we have incorporated many of the spectacularadvances I made over the past four years in biomedical science,driven in part by new experimental technologiesthat have revolutionized many fields. High-velocity techniquesfor sequencing DNA, for example,have generatedthe completesequence of dozens of eukaryotic genomes;these in turn have led to important discoveriesabout the organization of the human genome and regulation of gene expression,as well as novel insights into the evolution of life-forms and the functions of individual members of multiprotein families. New imaging techniqueshave generated profound revelations about cell organization and movement, and new molecular structures have greatly increased our understanding of life processes such as cell-cell signaling, photosynthesis, gene transcription, and chromatin structure.

what we know. A number of experimental organisms,from yeaststo worms to mice, are used throughout so the student can seehow discoveriesmade with a "lower organism" can Iead directly to insights even about human biology and disease.This experimental approach, evident in the text itself, has also been thoroughly integrated into the pedagogical framework. For example:

New Author Team

r Updated Perspectives for the Future essays explore potential applications of future discoveriesand unanswered questions that lie ahead in research.

Two new authors have been instrumental in refocusing this book toward these exciting new developments. Anthony Bretscher of Cornell University is known for identifying and characterizing new components of the actin cytoskeleton and elucidating their biological functions in relation to cell polarity and membrane traffic. Hidde Ploegh, of the Massachusetts Institute of Technologg has made major contributions to our understanding of immune system behavior, particularly in regard to the various tactics that viruses employ to evade our immune responsesand the ways our immune systemresponds. Both authors are widely recognized for their researchas well as their classroomteachingabilities. 'We are grateful to Paul Matsudaira, Jim Darnell, Larry ZipurskS and David Baltimore for their exceptional contributions to the previous editions of Molecular Cell Biology. Much of their vision and insight is apparent at many places in this book.

Experimental Emphasis The hallmark of Molecular Cell Biology has always been the use of experiments to teach students how we have learned

r Experimental Figures lead students through important experimental results. r Classic Experiments essaysfocus on historically important and Nobel Prize-winningexperiments. r New and revised Analyze the Data problems at the end of each chapter require the student to synthesizereal experimental data to answer a seriesof questions.

showsthe locationof DNAand multiple microscopy Fluorescence et al, 2006,Science BNG Giepmans proteins withinthe samecell.lFrom 3'12:217 |

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New Discoveries, New Methodologies

Coiled-coilstalk

Motor head Microtubule

Necklinkers

(+)

Methodological advances continue to expand and enrich our knowledge of molecular cell biology and lead to new understanding. Following are just a selection of the new experimental methodologiesand cutting-edgescienceintroduced in this edition: r Expanded coverage of proteomics, including organelle proteome profiling and advances in mass spectroscopy (Chapter 3)

I ForwardmotorbindsB-tubulin, ADP ^ releasing

p ForwardheadbindsATP

?

r Expanded coverageof RNAi, including the use of shRNAs to inhibit any gene of interest in a cultured cell or organism (Chapters5, 8) r Updated discussionsof chromatin, including structure and condensation(Chapter 6), control of geneexpressionby chromatin remodeling (Chapter 71, and chromatin-remodeling proteins and tumor development(Chapter 25) r Evolution (Chapter 6)

p

Conformationalchangein necklinkercausesrear head to swing forward

of chromosomes and the mitochondrion

r New molecular models, including pre-initiation complex and mediator complex (Chapter 7); annular phospholipids (Chapter 1,0);Caz* ATPase (Chapter 11); rhodopsin, transducin, and protein kinase A (Chapter 15); and myosin ATPase (Chapter 17) r Latest advancesin light and electron microscopy, including cryoelectron tomography (Chapter 9) r Reactive oxygen species(ROS) (Chapter 12)

ADB [ rue* forwardheadreleases ^ trailingheadhydrolyzesATP V and releases P.

r Role of supercomplexesin electron transport (Chapter 12) r Human epidermal growth factor receptors (HERs) and treatment of cancer (Chapter 16) r Myosin ATPasecycle (Chapter 17) r Kinesin-1MPase cycle (Chapter 18) r Use of retrovirus infection for tracing cell lineage (Chapter 21) r Axon guidance molecules (Chapter 23) r Somaticgenerearrangementin immune cells (Chapter 24) r Cancer stem cells (Chapter25) r Use of DNA (Chapter25)

Figure18-22 Kinesin-1 usesATPto "walk" downa microtubule

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PREFACE

microarray analysis in tumor typing

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For Students Companion Web Site www.whfreeman. com,/lodish5e NEW: Podcastsnarrated by the authors give students a deeperunderstanding of key figures in the text and a senseof the thrill of discovery. r NEW: Now available for your MP3 player or personal computer, more than 1,25animations and researchvideos show the dynamic nature of key cellular processesand important experimental techniques.The animations were storyboarded by the textbook authors in conjunction with BioStudio, Inc., and programmed by Sumanas,Inc. r Classic Experiment essaysfocus on classicgroundbreaking experiments and explore the investigativeprocess. r Online Quizzing is provided, including multiple-choice and short answer questions. -4), written Student SolutionsManual (ISBN:1-4292-01.27 'Wong, Richard Walker, Glenda by Brian Storrie, Eric A. Gillaspn and Jill Sible of Virginia Polytechnic Institute and State University and updated by Cindy Klevickis of James Madison University and Greg M. Kelly of the University of Western Ontario, contains complete workedout solutions to all the end-of-chapter problems in the textbook. NEW: eBook (ISBN: 1,-4292-0955-0)New to the sixth edition, this customizable eBook fully integrates the complete contents of the text and its interactive media in a format that features a variety of helpful study tools, including fulltext searching,note-taking, bookmarking, highlighting, and more. Easily accessibleon any Internet-connectedcomputer via a standard rWebbrowser, the eBook enablesstudents to take an active approach to their learning in an intuitive, easy-

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For Instructors Companion Web Site www.whfreeman.com/lodish6e All the student resources,plus: r All figures and tables from the book in ipeg and layered PowerPoint formats, which instructors can edit or project section by section, allowing students to follow underlying concepts.Optimized for lecture-hall presentation' including enhancedcolors, enlarged labels, and boldface rype. r Test Bank in editable Microsoft'Word format now featuringnetu and reuisedquestionsfor every chapter.The test bank is written by Brian Storrie of the University of Arkansas for Medical Sciencesand Eric A. Wong, Richard Walker, Glenda GillaspS and Jill Sible of Virginia Polytechnic Institute and StateUniversity and revisedby Cindy Klevickis of JamesMadison University and Greg M. Kelly of the University of Ontario. r Additional Analyze the Data problems are available in PDF format. r NEW: Lecture-ready Personal ResponseSystem "clicker" questions are available as Microsoft'Word files and Microsoft PowerPoint slides. Instructor's Resource CD-ROM (ISBN: 1'-4292-0126-6) includesall the instructor's resourcesfrom the Web site' including all the illustrations from the text, animations, videos, test bank files, clicker questions, and the solutions manual files. 'l'-4292-0477-X) conOverhead tansparency Set (ISBN: for optimized text' the from key illustrations tains 250 presentation. lecture-hall

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PREFACE

'

ACKNOWLEDGMENTS In updating, revising and rewriting this book, we were given 'We invaluable help by many colleagues. thank the following people who generouslygave of their time and expertiseby making contributions to specific chapters in their areas of interest, providing us with detailed information about their courses, or by reading and commenting on one or more chapters: Steven Ackerman, (Jniuersity of Massacbusetts,Boston Richard AdIe4 IJniuersity of Michigan, Dearborn Karen Aguirre, Coastal Carolina lJniuersity Jeff Bachant, Uniuersity of California, Riuerside Kenneth Balazovich, Uniuersity of Michigan Ben A. Barres, Stanford Uniuersity Karen K. Bernd, Dauidson College Sanford Bernstein, San Diego State (Jniuersity Doug Black, Howard Hughes Medical Institute and (Jniuersity of California, Los Angeles Richard L. Blanton, North Carolina State (Jniuersny Justin Blau, New York [Jniuersity Steven Block, Stanford IJniuersity Jonathan E. Boyson, Uniuersity of Vermont Janet Braam. Rice Uniuersity Roger Bradleg Montana State Uniuersity IilTilliam S. Bradshaq Brigham Young (Jniuersity Gregory G. Brown, McGill (Jniuersity \Tilliam J. Brown, Cornell IJniuersity Max M. Burger, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland David Burgess,Boston College Robin K. Cameron, McMaster [Jniuersity \7. Zacheus Cande, (Jniuersity of California, Berkeley Steven A. Carr, Broad lnstitute of Haruard (Jniuersity and Massacbusetts Institute of Technology Alice Y. Cheung, IJniuersity of Massacbusetts, Amberst Dennis O. Clegg, Uniuersity of California, Santa Barbara Paul Clifton, Utah State IJniuersity Randy \7. Cohen, California State [Jniuersity, Northridge Richard Dickerson, (Jniuersity of California, Los Angeles Patrick J. DiMario, Louisiana State Uniuersity Santosh R. D'Mello, [Jniuersity of Texas, Dallas Chris Doe, HHMI and [Jniuersity of Oregon Robert S. Dotson, Tulane [Jniuersity 'Sfilliam Dowhan, IJniuersity of Texas-Houston Medical School Gerald B. Downes, (Jniuersity of Massachusetts,Amherst Erastus C. Dudley, Huntingdon College Susan Dutcher, V/ashington (Jniuersity School of Medicine Matt Elrod-Erickson, Middle TennesseeState Uniuersity Susan Ely, Cornell Uniuersity Charles P. Emerson Jr., Boston Biomedical ResearchInstitute Irene M. Evans, Rochester Institute of Technology James G. Evans,.Whitehead Institute Bio Imaging Center, Massachusetts Institute of Technology Marilyn Gist Farquhar, Uniuersity of California, San Diego

X

PREFACE

Xavier Fernandez-Busquets, Bioengineering Instituteof Catalonia, Uniuersitatde Barcelona,Spain TerrenceG. Frey,San Diego State Uniuersity Margaret T. Fuller,Stanford UniuersitySchoolof Medicine KendraJ. Golden, WbitmanCollege David S. Goldfarb,RochesterUniuersity Martha J. Grossel,ConnecticutCollege LawrenceI. Grossman,WayneStateUniuersitySchoolof Medicine Michael Grunstein, Uniuersityof California, Los Angeles, Schoolof Medicine Barry M. Gumbiner, Uniuersityof Virginia 'Wei Guo, Uniuersityof Pennsyluarua Leah Haimo, Uniuersityof California, Riuerside Heidi E. Hamm, Vanderbilt(JniuersityMedicalSchool Craig M. Hart, Louisiana State Uniuersity Merill B. Hille, Uniuersityof Washington Jerry E. Honts, Drake Uniuersity H. Robert Horvitz, Massachusetts Institute of Technology Richard Hynes, Massachusetts lnstitute of Technologyand Howard HughesMedical Inshtute Harry Itagaki,Kenyon College ElizabethR. Jamieson,Smitb College Marie A. Janicke,State Uniuersityof New York, Buffalo Bradley'W.Jones,Uniuersityof Mississippi Mark Kainz, ColgateUniuersity Naohiro Kato, Louisiana State Uniuersity Amy E. Keating, Massachusetts lnstitute of Technology CharlesH. Keith, Uniuersityof Georgia Thomas C. S. Keller lll, Florida State Uniuersity 'V/estern Greg M. Kelly, Uniuersityof Ontario StephenKendall, California State Uniuersity,Fullerton FelipeKierszenbaum, MichiganStateUniuersity Cindy Klevickis,JamesMadison lJniuersity Brian Kobilka, StanfordUniuersityMedicalSchool. Martina Koniger, WellesleyUniuersfiy CatherineKoo, Caldwell College Keith G. Kozminski, Uniuersityof Virginia StevenI(. IJHernault, Emory Uruuerstty Douglas Lauffenburger, Massacbusetts Institute of Tecbnology RobertJ. Lefkowitz, HHMI and Duke UniuersityMedical School R. L. Levine,McGill Uniuersity FangJu Ltn, CoastalCarolina [Jniuersity ElizabethLord, Uniuersityof California, Riuerside Liqun Luo, StanfordUniuersity Grant MacGregor, Uniuersityof California, Iruine Jennifer O. Manilay, IJniuersityof California, Merced Barry Margul ies,Towson Uniuer stty C. William McCurdy, Uniuersityof California, Dauis, and LawrenceBerkeleyNational Laboratory Dennis \il/. McGee, State Uniuersityof New York, Binghamton JamesMcGrath, RochesterSchoolof Medicine David D. McKemy, Uniuersityof SouthernCalifornia Roderick MacKinnon, Rockefeller(Jniuersity JamesA. McNew, Rice Uniuersity

X

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CONTENTS IN BRIEF

Part I 1. 2. 3. Part ll 4. 5. 6. 7. 8. Part lll 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Chemical and MolecularFoundations1 L i f e B e g i n sw i th C e l l s 1 C h e mi caFl o u n d a ti o n s3 1 ProteinStructureand Function 63

Genetics and MolecularBiology 111 B a s i cM o l e c u l aG r e n e t i cM e c h a n i s m1s 1 1 M o l e cu l a Ge r n e ti cT e ch n i ques1G5 G e n e s,Ge n o mi cs, a n d C h rom osomes 215 Transcriptional Controlof Gene Expression269 Post-transcriptional GeneControl 323

CeflStructureand Function 371 V i su a l i zi n gF, ra cti o n a ti n g, and Cultur ingCells 371 BiomembraneStructure 409 Transmembrane Transportof lonsand small Molecures437 C e ll u l a rE n e rg e ti cs4 7 9 M o vi n gP ro te i n si n to Me mbr anes and Or ganelles533 VesicularTraffic,Secretion,and Endocytosis579 C e llS i g n a l i n gl : S i g n a T l ra nsduction and Shor t- Ter m CellularResponses 62 3 c e l l si g n a l i n gl l : si g n a l i n gPathways That contr ol GeneActivity 665 C e l lOrg a n i za ti o n a n d Mo v ementl: M icr ofilaments713 C e l lOrg a n i za ti o n a n d Mo v ementll: Micr otubules and Inter m ediate Filaments157 I n t e g ra ti n gC e l l si n to T i ssues801

Part lV CellGrowth and Development847 20. Regulatingthe EukaryoticCellCycle 847 21. 22. 23. 24. 25.

C e l lB i rth ,L i n e a g ea, n d D e ath 905 T h e Mo l e cu l a C r e l lB i o l o g yof Developm entg4g N e rveC e l l s 1 0 0 1 l m m u n o l o g y1 0 5 5 Cancer 1107

CONTENTS

PartI Chemical and MolecularFoundations

E

The Diversityand Commonality of Cells

18

C e l l sG r o w a n d D i v i d e

WITHCELLS 1 L I F EB E G I N S

CellsDie from AggravatedAssaultor an Internal Program 19

1

!!|

InvestigatingCellsand Their Parts 20

All CellsAre Prokaryoticor Eukaryotic

1

U n i c e l l u l aO r r g a n i s m sH e l p a n d H u r t U s

4

VirusesAre the Ultimate Parasites

6

Cell BiologyRevealsthe Size,Shape,Location, 20 and Movementsof Cell Components Biochemistryand BiophysicsRevealthe Molecular Structureand Chemistryof PurifiedCell Constituents 21

C h a n g e si n C e l l sU n d e r l i eE v o l u t i o n

6

of DamagedGenes 22 GeneticsRevealsthe Consequences

EvenSingleCellsCan HaveSex

7

We Developfrom a SingleCell

8

GenomicsRevealsDifferencesin the Structure of EntireGenomes and Expression

23

DevelopmentalBiologyRevealsChangesin the Propertiesof Cellsas They Specialize

23

Stem Cells,Fundamentalto FormingTissues and Organs,Offer MedicalOpportunities

@The

Choosingthe Right ExperimentalOrganismfor the Job 25 BiologicalStudiesUse Multiple The Most Successful 27 Approaches

M o l e c u l eos f a c e l l

Small MoleculesCarryEnergy,TransmitSignals, a n d A r e L i n k e di n t o M a c r o m o l e c u l e s ProteinsGive CellsStructureand Perform Most CellularTasks

'

s|

on Evolution 28 A GenomePerspective

10

Metabolic Proteins,the GeneticCode,and Organelle StructuresAre NearlYUniversal

't1

The Genome ls Packagedinto Chromosomesand R e p l i c a t e dD u r i n gC e l lD i v i s i o n

Darwin'sldeasAbout the Evolutionof Whole Animals Are Relevantto Genes

12

Mutations May Be Good, Bad, or lndifferent

13

Many GenesControllingDevelopmentAre Remarkably 28 S i m i l a ri n H u m a n sa n d O t h e r A n i m a l s Human Medicinels Informed by Researchon Other 29 Organisms

NucleicAcidsCarryCoded Information for Making Proteinsat the Right Time and Place

E

Thework of cells

28

14

C e l l sB u i l da n d D e g r a d eN u m e r o u sM o l e c u l e s and Structures

15

Animal CellsProduceTheir Own External E n v i r o n m e nat n d G l u e s

16

CellsChangeShapeand Move

16

CellsSenseand Send Information

16

covalent Bondsand Noncovalent lnteractions

to CellsRegulateTheir Gene Expression M e e t C h a n g i n gN e e d s

17

the Structureof an Atom Determines TheElectronic of CovalentBondslt CanMake 33 Numberand Geometry

31

FOUNDATIONS 2 CHEMICAL ![

CONTENTS

.

32

E l e c t r o n sM a y B e S h a r e dE q u a l l yo r U n e q u a l l y i n C o v a l e n tB o n d s

34

L i f e D e p e n d so n t h e C o u p l i n go f U n f a v o r a b l eC h e m i c a l Reactionswith EnergeticallyFavorableReactions 57

CovalentBondsAre Much Strongerand More StableThan NoncovalentInteractions

Hydrolysisof ATPReleases SubstantialFreeEnergy and DrivesMany CellularProcesses

lonic InteractionsAre Attractionsbetween Oppositely C h a r g e dl o n s 36 HydrogenBondsDeterminethe Water Solubility o f U n c h a r g e dM o l e c u l e s 37

ATPls GeneratedDuring Photosynthesis and Respiration 59 N A D - a n d F A DC o u p l eM a n y B i o l o g i c aO l xidation and ReductionReactions

Van der Waals InteractionsAre Causedby T r a n s i e nD t iooles

37

The HydrophobicEffectCausesNonpolar Moleculesto Adhere to One Another

38

3 P R O T E ISNT R U C T U R ED AN FUNCTION

M o l e c u l a rC o m p l e m e n t a r i tM y e d i a t e dv i a NoncovalentInteractionspermitsTight, H i g h l yS p e c i f i cB i n d i n go f B i o m o l e c u l e s

3e

![

C h e m i c alB u i l d i n gB l o ckso f C e i l s

@

A m i n o A c i d sD i f f e r i n gO n l y i n T h e i rS i d eC h a i n s ComposeProteins

40 41

FiveDifferent NucleotidesAre Usedto Build N u c l e i cA c i d s Monosaccharides Joined by GlycosidicBonds Form Linearand Branchedpolysaccharides

44

PhospholipidsAssociateNoncovalentlyto Form the BasicBilayerStructureof Biomembranes

C h e m i c aEl q u i l i b ri u m

49

EquilibriumConstantsReflectthe Extentof a ChemicalReaction

50

![

Hierarchical Structureof proteins

SecondaryStructuresAre the Core Elements of ProteinArchitecture

66

Overall Foldingof a PolypeptideChainYields Its TertiaryStructure

67

Different Waysof Depictingthe Conformationof ProteinsConveyDifferent Typesof Information

68

StructuralMotifs Are RegularCombinationsof Secondaryand TertiaryStructures

68

S t r u c t u r aal n d F u n c t i o n aD l o m a i n sA r e M o d u l e s of TertiaryStructure

70

ProteinsAssociateinto Multimeric Structuresand M a c r o m o l e c u l aAr s s e m b l i e s

72

Membersof Protein FamiliesHavea Common EvolutionaryAncestor

72

![

BiologicalFluidsHaveCharacteristic pH Values

51

P l a n a rP e p t i d eB o n d sL i m i tt h e S h a p e si n t o Which ProteinsCan Fold

B i o c h e m i caEl n e rg e ti cs

@

SeveralFormsof EnergyAre lmportant in BiologicalSystems CellsCan TransformOne Typeof Energyinto Another The Changein Free EnergyDeterminesthe Direction of a ChemicalReaction The AG'' of a ReactionCan Be Calculated from lts K"o The Rateof a ReactionDependson the Activation EnergyNecessary to Energizethe Reactantsinto a TransitionState

xvi

.

coNTENTs

64 65

50

B u f f e r sM a i n t a i nt h e p H o f I n t r a c e l l u l aar n d E x t r a c e l l u l aFrl u i d s

63

The PrimaryStructureof a Protein ls lts Linear Arrangementof Amino Acids

ChemicalReactionsin CellsAre at SteadyState DissociationConstantsof Binding ReactionsReflect the Affinity of InteractingMolecules Hydrogenlons Are Releasedby Acidsand Taken Up by Bases

57

50

52 52

ProteinFolding

74 74

I n f o r m a t i o nD i r e c t i n ga P r o t e i n ' sF o l d i n gl s E n c o d e di n l t s A m i n o A c i d S e q u e n c e

74 Foldingof Proteinsin Vivo ls Promotedby Chaperones 7 5 AlternativelyFoldedProteinsAre lmplicatedin Diseases 77

54

ProteinFunction

78

S p e c i f i cB i n d i n go f L i g a n d sU n d e r l i e st h e Functionsof Most Proteins

7g

EnzymesAre Highly Efficientand SpecificCatalysts

79

An Enzyme'sActive Site BindsSubstratesand CarriesOut Catalysis

g0

SerineProteasesDemonstrateHow an Enzyme'sActive Site Works

g1

Enzymesin a Common PathwayAre Often physically Associatedwith One Another

g4

!f, 54 55 55 56

56

EnzymesCalledMolecularMotors ConvertEnergy into Motion

!E-

!l|

r.r RegulatingProteinFunctionl: ProteinDegradation

RegulatedSynthesisand Degradationof Proteins is a FundamentalPropertyof Cells

CentrifugationCan SeparateParticlesand MoleculesThat Differ in Massor Density

86 86

The Proteasomels a ComplexMolecularMachine Usedto DegradeProteins Ubiquitin Marks CytosolicProteinsfor Degradation in Proteasomes

RegulatingProteinFunctionll: Noncovalentand Covalent Modifications NoncovalentBinding PermitsAllosteric, or Cooperative,Regulationof Proteins

Purifying,Detecting,and Proteins Characterizing

85

SeparatesMoleculeson the Basis Electrophoresis Ratio of Their Charge-to-Mass Liquid ChromatographyResolvesProteinsby Mass,Charge,or BindingAffinitY

92 92 94 96

Highly SpecificEnzymeand Antibody AssaysCan Detect IndividualProteins Toolsfor Detecting Are Indispensable Radioisotopes M o l e c u l e s Biological MassSpectrometryCan Determinethe Mass and Sequenceof Proteins

101

88

Protein PrimaryStructureCan Be Determinedby ChemicalMethods and from Gene Sequences

103

89

Protein Conformationls Determinedby Sophisticated 103 PhysicalMethods

88

NoncovalentBindingof Calciumand GTPAre Widely UsedAs AllostericSwitchesto Control ProteinActivity 90 Phosphorylationand DephosphorylationCovalently RegulateProteinActivitY

91

ProteolyticCleavagelrreversiblyActivatesor lnactivatesSome Proteins

91

Higher-OrderRegulationIncludesControl of Protein Locationand Concentration

92

g

98 99

105

Proteomics

Proteomicsls the Studyof All or a LargeSubset of Proteinsin a BiologicalSYstem AdvancedTechniquesin MassSpectrometryAre Criticalto ProteomicAnalYsis

105 106

Partll Geneticsand MolecularBiologY 4 B A S I CM O L E C U L AGRE N E T I C MECHANISMS @

structureof NucleicAcids

111 113

Organizationof GenesDiffersin Prokaryoticand EukaryoticDNA to EukaryoticPrecursormRNAsAre Processed l RNAs F o r mF u n c t i o n am the Number of AlternativeRNA Splicinglncreases from a SingleEukaryoticGene ProteinsExpressed

A NucleicAcid Strand ls a LinearPolymerwith End-to-EndDirectionality

113

@

N a t i v eD N A l s a D o u b l eH e l i xo f C o m p l e m e n t a r y A n t i p a r a l l eS l trands

114

DNA Can Undergo ReversibleStrandSeparation

115

MessengerRNA CarriesInformation from DNA in a Three-LetterGeneticCode The FoldedStructureof IRNA Promoteslts Decoding

TorsionalStressin DNA ls Relievedby Enzymes

117

Different Typesof RNA ExhibitVarious ConformationsRelatedto Their Functions

118

@ T r a n s c r i p t i o n o f P ro te i n -C o d i ng Genesand Formationof Functional 120 MRNA A TemplateDNA Strand ls Transcribedinto a ComplementaryRNA Chain by RNA Polymerase

120

122 123 125

The Decodingof mRNAbY tRNAs 127

Functions NonstandardBasePairingOften OccursBetween C o d o n sa n d A n t i c o d o n s Amino Acids BecomeActivatedWhen Covalently L i n k e dt o t R N A s

@

StepwiseSynthesisof Proteins on Rabosomes

Machines RibosomesAre Protein-Synthesizing

CONTENTS

127 129 130 131

132 132

XVII

Methionyl-tRNA,tttRecognizes the AUG StartCodon T r a n s l a t i o nI n i t i a t i o nU s u a l l yO c c u r sa t t h e F i r s t A U G f r o m t h e 5 ' E n do f a n m R N A D u r i n gC h a i nE l o n g a t i o nE a c hl n c o m i n g Aminoacyl-tRNAMovesThrough Three RibosomalSites Translationls Terminatedby ReleaseFactors When a Stop Codon ls Reached Polysomesand RapidRibosomeRecyclingIncrease the Efficiencyof Translation

DNAReptication

!f|

133 133

E! 135

166

137

166

138

Segregationof Mutations in BreedingExperiments RevealsTheir Dominanceor Recessivity

167

ConditionalMutations Can Be Usedto Study EssentialGenesin Yeast

EO

139 140

Duplex DNA ls Unwound and Daughter StrandsAre Formedat the DNA ReplicationFork

't41 141 143

Recessive Lethal Mutations in DiploidsCan Be l d e n t i f i e db y I n b r e e d i n ga n d M a i n t a i n e di n Heterozygotes

171 ComplementationTestsDetermineWhether Different 't7 Recessive Mutations Are in the SameGene 1 Double Mutants Are Usefulin Assessing the Order in Which ProteinsFunction 171 GeneticSuppression and SyntheticLethalityCan RevealInteractingor Redundantproteins 173 GenesCan Be ldentified by Their Map position on the Chromosome 174

D N A R e p a i ra n d R e co mb i n a ti o n 145

!!|

p|

DNA Polymerases IntroduceCopyingErrors and Also CorrectThem

145

C h e m i c aal n d R a d i a t i o nD a m a g et o D N A C a n Leadto Mutations

145

H i g h - F i d e l i tD y N A E x c i s i o n - R e p aSiyr s t e m s Recognize a n d R e p a i rD a m a g e B a s eE x c i s i o n R e p a i r sT . G M i s m a t c h eas n d D a m a g e dB a s e s

147

MismatchExcisionRepairsOther Mismatchesand S m a l lI n s e r t i o n a s nd Deletions

147

NucleotideExcisionRepairsChemicalAdducts That Distort Normal DNA Shape Two SystemsUtilize Recombinationto Repair D o u b l e - S t r a nB d r e a k si n D N A H o m o l o g o u sR e c o m b i n a t i oC n a n R e o a i rD N A D a m a g ea n d G e n e r a t eG e n e t i cD i v e r s i t y

s f th e C e l l u l a r @ V i r u s e s : pa ra si te o GeneticSystem Most Viral Host RangesAre Narrow Viral CapsidsAre RegularArraysof One or a Few Typesof protein

147

148 149 150

154

'

CONTENTS

DNACloningand Char acter i z ati on176

RestrictionEnzymesand DNA LigasesAllow Insertionof DNA Fragmentsinto CloningVectors

176

E. coli PlasmidVectorsAre Suitablefor Cloning lsolatedDNA Fragments

178

cDNA LibrariesRepresentthe Sequences of Protein-CodingGenes

179 cDNAsPreparedby ReverseTranscriptionof Cellular mRNAsCan Be Clonedto GeneratecDNA Libraries 1 8 1 DNA LibrariesCan Be Screenedby Hybridization to an Oligonucleotidp erobe 181 YeastGenomicLibrariesCan Be Constructedwith ShuttleVectorsand Screenedby Functionar Complementation 182 Gel Electrophoresis Allows Separationof Vector DNA from Cloned Fragments 184 Cloned DNA MoleculesAre SequencedRapidly by the DideoxyChain-Termination Methoc 187 The PolymeraseChain ReactionAmplifiesa Specific DNA Sequencefrom a ComplexMixture 188

154 154

VirusesCan Be Clonedand Counted in plaqueAssays 1 5 5 LyticViral Growth CyclesLeadto the Death of Host Cells 1 5 5 Viral DNA ls Integrated into the Host-CellGenome in SomeNonlyticViral Growth Cycles 158

XVIII

GeneticAnalysisof Mutationsto ldentifyand StudyGenes

R e c e s s i vaen d D o m i n a n tM u t a n t A l l e l e sG e n e r a l l y HaveOppositeEffectson Gene Function

D N A P o l y m e r a s eR s e q u i r ea p r i m e r t o I n i t i a t e Replication

SeveralProteinsParticipatein DNA Replication D N A R e p l i c a t i o nU s u a l l yO c c u r sB i d i r e c t i o n a l lfyr o m E a c hO r i g i n

5 M O L E C U L AGRE N E T T C E C H N T Q U1E6Ss

ff|

UsingClonedDNA Fragments to Study GeneExpression

191

HybridizationTechniquespermit Detection of SpecificDNA Fragmentsand mRNAs

't91

DNA MicroarraysCan Be Usedto Evaluatethe Expression of Many Genesat One Time

192

ClusterAnalysisof Multiple ExpressionExperiments ldentifies Co-regulatedGenes

193

N o n p r o t e i n - C o d i nG g e n e sE n c o d eF u n c t i o n a l RNAs

SystemsCan ProduceLarge E. coli Expression Quantitiesof Proteinsfrom ClonedGenes VectorsCan Be Designedfor PlasmidExpression U s ei n A n i m a l C e l l s

Or ganization Chr om osomal of Genesand NoncodingDNA

196

222

223

s o n t a i nM u c h G e n o m e so f M a n y O r g a n i s m C N o n f u n c t i o n aD l NA

l d e n t i f y in ga n d L o ca ti n gH u ma n Disease Ge n e s

DNAsAre Concentrated Most Simple-Sequence in SpecificChromosomalLocations

224

199

DNA FingerprintingDependson Differences DNAs in Length of SimPle-Sequence

225

D N A P o l y m o r p h i s mAsr e U s e di n L i n k a g e - M a p p i n g H u m a nM u t a t i o n s

200

SpacerDNA Occupiesa Significant Unclassified Portion of the Genome

225

LinkageStudiesCan Map DiseaseGeneswith a Resolutionof About 1 Centimorgan

201

FurtherAnalysisls Neededto Locatea DiseaseGene i n C l o n e dD N A

202

f[

Show One of Three Major Many InheritedDiseases Patternsof Inheritance

198

]f|

M a n y I n h e r i t e dD i s e a s eRs e s u l ft r o m M u l t i p l eG e n e t i c 203 Defects

ffl

Inactivatingthe Functionof S p e c i f i cGe n e si n E u ka ryo te s

Normal YeastGenesCan Be Replacedwith Mutant A l l e l e sb y H o m o l o g o u sR e c o m b i n a t i o n

204 205

Transcriptionof GenesLigatedto a Regulated PromoterCan Be ControlledExperimentally SpecificGenesCan Be PermanentlyInactivatedin t h e G e r m L i n eo f M i c e

207

SomaticCell RecombinationCan InactivateGenes in SpecificTissues

208

D o m i n a n t - N e g a t i vAel l e l e sC a n F u n c t i o n a l l y l n h i b i t S o m eG e n e s RNA InterferenceCausesGene Inactivationby g RNA D e s t r o y i n gt h e C o r r e s p o n d i nm

6 G E N E SG , E N O M I C SA, N D CHROMOSOMES

][

EukaryoticGeneStructure

(Mobile)DNA Transposable Elem ents

Movement of Mobile ElementsInvolvesa D N A o r a n R N AI n t e r m e d i a t e

226

Are Presentin Prokaryotes DNA Transposons and Eukaryotes

227

BehaveLike Intracellular LTRRetrotransposons Retroviruses Transposeby a Distinct Non-LTRRetrotransposons Mechanism RNAsAre Found in Genomic Other Retrotransposed DNA M o b i l e D N A E l e m e n t sH a v eS i g n i f i c a n t l Iyn f l u e n c e d Evolution

@

215 217

Most EukaryoticGenesContain Intronsand Produce m R N A sE n c o d i n gS i n g l eP r o t e i n s

217

n nits S i m p l ea n d C o m p l e xT r a n s c r i p t i oU A r e F o u n di n E u k a r y o t i cG e n o m e s

217

P r o t e i n - C o d i nG g e n e sM a y B e S o l i t a r yo r B e l o n g t o a G e n eF a m i l y

219

HeavilyUsedGene ProductsAre Encodedby Multiple 221 Copiesof Genes

229 230 234 234

236

or ganelleDNAs

M i t o c h o n d r i aC o n t a i nM u l t i p l e m t D N A M o l e c u l e s

210

226

mtDNA ls InheritedCytoplasmically The Size,Structure,and Coding Capacityof mtDNA Vary ConsiderablyBetweenOrganisms Productsof MitochondrialGenesAre Not Exported M i t o c h o n d r i aE v o l v e df r o m a S i n g l eE n d o s y m b i o t i c Bacterium EventInvolvinga Rickettsia-like

237 237 238 240

240

MitochondrialGeneticCodesDiffer from the StandardNuclearCode

240

Mutations in MitochondrialDNA CauseSeveral G e n e t i cD i s e a s eisn H u m a n s ChloroplastsContain LargeDNAsOften Encoding M o r e T h a n a H u n d r e dP r o t e i n s

242

Anal Ys i s Genome- wide Genom ics: of GeneStructureand Expression 243 SuggestFunctionsof Newly StoredSequences ldentified Genesand Proteins

CONTENTS

243

xtx

Comparisonof RelatedSequences from Different SpeciesCan Give Cluesto Evolutionary Relationships Among proteins G e n e sC a n B e l d e n t i f i e dW i t h i n G e n o m i cD N A Sequences

244

S m a l lM o l e c u l e sR e g u l a t eE x p r e s s i oonf M a n y BacterialGenesvia DNA-BindingRepressors and Activators

273

244

TranscriptionInitiation from Some Promoters RequiresAlternativeSigmaFactors

273

Transcriptionby osa-RNAPolymerasels Controlled by ActivatorsThat Bind Farfrom the Promoter

274

Many BacterialResponses Are Controlledby Two-ComponentRegulatorySystems

275

T h e N u m b e ro f P r o t e i n - C o d i nG g e n e si n a n Organism'G s e n o m el s N o t D i r e c t l yR e l a t e d to lts BiologicalComplexity S i n g l eN u c l e o t i d eP o l y m o r p h i s masn d G e n eC o p y Number VariationAre lmportant Determinants of DifferencesBetween Individualsof a Species

246

@ St r u c t u r alOrg a n i za ti o n of EukaryoticChromosomes

][

ChromatinExistsin Extendedand CondensedForms Modificationsof HistoneTailsControl Chromatin Condensationand Function NonhistoneProteinsProvidea StructuralScaffold for Long ChromatinLoops A d d i t i o n a lN o n h i s t o n ep r o t e i n sR e g u l a t e Transcriptionand Replication

247 248

276

250

Three EukaryoticPolymerases CatalyzeFormation of Different RNAs

278

254

The LargestSubunit in RNA Polymerasell Hasan EssentialCarboxyl-Terminal Repeat

2s6

ChromosomeNumber;Size,and Shapeat Metaphase Are Species-Specific 257 During Metaphase,ChromosomesCan Be D i s t i n g u i s h ebdy B a n d i n gp a t t e r n sa n d C h r o m o s o m eP a i n t i n g 25g C h r o m o s o m eP a i n t i n ga n d D N A S e q u e n c i n g Reveal the Evolutionof Chromosomes 259 InterphasePolyteneChromosomesArise by DNA A m p l i fi c a t i o n 260 Three FunctionalElementsAre Requiredfor Replication a n d S t a b l eI n h e r i t a n c eo f C h r o m o s o m e s 261 CentromereSequences Vary Greatlyin Length 263 Addition of TelomericSequencesby Telomerase PreventsShorteningof Chromosomes

263

7 TRANSCRIPTIONALCONTROL O F G E N EE X P R E S S I O N 269 Controlof GeneExpression in Bacteria

W

271

TranscriptionInitiation by BacterialRNA polymerase RequiresAssociationwith a SigmaFactor 271 Initiation of /ac Operon TranscriptionCan Be Repressed and Activated 271

XX

O

CONTENTS

276

RegulatoryElementsin EukaryoticDNA Are Found Both Closeto and Many KilobasesAway from TranscriptionStart Sites

M o r p h o l og ya n d F u n cti o n aEl l e m ents o f E u k a r y o ti C c h ro mo so me s 257

@

overview of EukaryoticGene Controland RNAPolymerases

279 RNA Polymerasell InitiatesTranscriptionat DNA SequencesCorrespondingto the 5' Cap of mRNAs 280

E

Regulator ysequences in pr ote i nCodingGenes 282

The TATABox, Initiators,and CpG lslandsFunction as Promotersin EukaryoticDNA

282

Promoter-Proximal ElementsHelp Regulate EukaryoticGenes

282

Distant EnhancersOften Stimu late Transcription by RNA Polymerasell

284

Most EukaryoticGenesAre Regulatedby Multiple Transcription-Control Elements

s @Activator s and Repr essorof Transcription

286

Footprintingand Gel-ShiftAssaysDetect protein-DNA Interactions 286 ActivatorsAre Modular ProteinsComposed of DistinctFunctionalDomainsand promote Transcription 288 Repressors Inhibit Transcriptionand Are the FunctionalConverseof Activators

290

D N A - B i n d i n gD o m a i n sC a n B e C l a s s i f i eidn t o NumerousStructuralTypes

290

StructurallyDiverseActivation and Repression DomainsRegulateTranscription

293

TranscriptionFactorInteractionsIncrease Gene-ControlOptions

294

M u l t i p r o t e i nC o m p l e x e F s o r mo n E n h a n c e r s

295

TranscriptionInitiation by RNA Polymerasell GeneralTranscriptionFactorsPositionRNA Polymerasell at Start Sitesand Assistin Initiation

296

@

GENE 8 POST- TRANSCRIPTIONAL CONTROL

Formationof HeterochromatinSilencesGene at Telomeres,Near Centromeres, Expression and in Other Regions

299

Can Direct HistoneDeacetylationand Repressors Methylation at SpecificGenes

303

ActivatorsCan Direct HistoneAcetylationand Methylation at SpecificGenes

305

FactorsHelp Activate or Chromatin-Remodeling RepressTranscription

306

HistoneModificationsVary Greatlyin Their Stabilities 307 307

Transcriptionof Many GenesRequiresOrdered Binding 308 and Functionof Activatorsand Co-activators The YeastTwo-HybridSystemExploitsActivator Flexibility to DetectcDNAsThat EncodeInteractingProteins 310

E

312

ResponseElementsContain Inverted Nuclear-Receptor 313 or Direct Repeats Hormone Bindingto a NuclearReceptorRegulateslts 313 Activity as a TranscriptionFactor

![

RegulatedElongationand Terminationof Transcription

314

Transcriptionof the HIV Genome ls Regulatedby an Antitermination Mechanism

315

Pausingof RNA Polymerasell Promoter-Proximal Occursin Some RapidlyInducedGenes

316

@

f[

other EukaryoticTranscription Systems

TranscriptionInitiation by Pol I and Pol lll ls Analogousto That by Pol ll

315 316

of EukaryoticPre-mRNA325 Processing

The 5' Cap ls Added to NascentRNAsShortlyAfter TranscriptionInitiation

325

A DiverseSet of Proteinswith ConservedRNA-B|nding 326 DomainsAssociatewith Pre-mRNAs SplicingOccursat Short,ConservedSequencesin Pre-mRNAsvia Two TransesterificationReactions

329 330

with Pre-mRNA During Splicing,snRNAsBase-Pair Assembledfrom snRNPsand a Spliceosomes, Pre-mRNA,CarryOut SPlicing

330

C h a i nE l o n g a t i o nb y R N AP o l y m e r a slel l s C o u p l e d Factors to the Presenceof RNA-Processing

333

SRProteinsContributeto Exon Definition in Long Pre-mRNAs Group ll Introns ProvideCluesto the Self-Splicing Evolutionof snRNAs

334

3' Cleavageand Polyadenylationof Pre-mRNAs 335 Are Tightly CouPled DegradeRNAThat ls Processed NuclearExonucleases 336 Out of Pre-mRNAs

Regulationof Transcription-Factor 311 Activity

All NuclearReceptorsSharea Common Domain Structure

323

298

o f T ra n sc r iption M o l e c u l a Me r ch a n i sms 299 and Activation Repression

The Mediator ComplexFormsa MolecularBridge BetweenActivation Domainsand Pol ll

317

296

SequentialAssemblyof ProteinsFormsthe Pol ll TranscriptionPreinitiationComplexin Vitro In Vivo TranscriptionInitiation by Pol ll Requires Additional Proteins

Mitochondrialand ChloroplastDNAsAre RNA Transcribedby Organelle-Specific Polymerases

Regulation of Pre-mRNA Processing

337

AlternativeSplicingls the PrimaryMechanismfor RegulatingmRNA Processing

337

A Cascadeof RegulatedRNA SplicingControls DrosophilaSexualDifferentiation

338

and ActivatorsControl Splicing SplicingRepressors at AlternativeSites

339

of RNA Editing Alters the Sequences SomePre-mRNAs

340

]f|

Transportof mRNAAcrossthe NuclearEnveloPe

341

NuclearPore ComplexesControl lmport and Export 342 from the Nucleus Are Not Exportedfrom in Spliceosomes Pre-mRNAs 345 the Nucleus HIV Rev Protein Regulatesthe Transportof Unspliced 346 Viral mRNAs

CONTENTS

'

xxi

CytoplasmicMechanisms of posttranscriptionalControl 347

f!|

Micro RNAsRepressTranslationof SpecificmRNAs RNA InterferenceInducesDegradationof precisely ComplementarymRNAs CytoplasmicPolyadenylationpromotesTranslation of SomemRNAs

Localizationof mRNAsPermitsProductionof Proteinsat SpecificRegionsWithin the Cytoplasm

357

347

Processing of rRNAand IRNA

358

349

f[

351

Pre-rRNAGenesFunctionas NucleolarOrganizers a n d A r e S i m i l a ri n A l l E u k a r y o t e s

359

Small NucleolarRNAsAssistin Processing Pre-rRNAs

360 363

Degradationof mRNAsin the CytoplasmOccurs by SeveralMechanisms

352

ProteinSynthesisCan Be GloballyRegulated

353

Self-Splicing Group I IntronsWere the First Examplesof CatalyticRNA

356

Pre-tRNAsUndergo ExtensiveModification in the Nucleus

363

357

N u c l e a rB o d i e sA r e F u n c t i o n a l lS y p e c i a l i z eN d uclear Domains

3G4

Sequence-Specific RNA-BindingproteinsControl SpecificmRNATranslation SurveillanceMechanismspreventTranslationof lmproperlyProcessed mRNAs

Partlll CellStructureand Function 9 V I S U A L I Z IN G, F R A C T ION A T IN G, A N D C U L T U R I NC GE L L S 371 Organellesof the EukaryoticCell

lll

372

Phase-Contrast and DifferentiallnterferenceContrast MicroscopyVisualizeUnstainedLivingCells 381 Fluorescence MicroscopyCan Localizeand euantify S p e c i f i cM o l e c u l e si n L i v eC e l l s 382 l m a g i n gS u b c e l l u l aDr e t a i l sO f t e n R e q u i r e tsh a t t h e SamplesBe Fixed,Sectioned,and Stained

T h e P l a s m aM e m b r a n eH a sM a n y C o m m o nF u n c t i o n s in All Cells 372 EndosomesTake Up 5oluble Macromolecules from the Cell Exterior 372

lmmunofluorescence MicroscopyCan Detect Specific Proteinsin FixedCells

38s

Confocaland DeconvolutionMicroscopyEnable Visualizationof Three-Dimensional Objects

386

Lysosomes Are Acidic OrganellesThat Contain a Batteryof DegradativeEnzymes

Graphicsand lnformaticsHaveTransformed Modern Microscopy

373

Peroxisomes DegradeFattyAcidsand ToxicCompounds 374 The EndoplasmicReticulumls a Network of InterconnectedInternal Membranes 375 The Golgi ComplexProcesses and SortsSecreted a n d M e m b r a n ep r o t e i n s

376

P l a n tV a c u o l e sS t o r eS m a l lM o l e c u l e sa n d E n a b l ea C e l lt o E l o n g a t eR a p i d l y

Eil

ElectronMicroscopy: Methods and Applications

388

Resolutionof Transmission ElectronMicroscopyis VastlyGreaterThan That of Light Microscopy

3gg

377

T h e N u c l e u sC o n t a i n st h e D N A G e n o m e ,R N A SyntheticApparatus,and a FibrousMatrix

CryoelectronMicroscopyAllows Visualizationof ParticlesWithout Fixationor Staining

399

378

MitochondriaAre the PrincipalSitesof ATp Productionin Aerobic NonphotosyntheticCells

378

ElectronMicroscopyof Metal-CoatedSpecimens Can RevealSurfaceFeaturesof Cellsand Their Components

390

ChloroplastsContain Internal Compartmentsin Which Photosynthesis Takesplace

379

!!| g

L i g h t M i cro sco p y: vi su a l i zi n gC ell Structureand Localizingproteins W i t h i n C el l s 380

The Resolutionof the Light Microscopels About 0.2 pm

xxii

.

coNTENrs

381

Purificationof Cell Organelles

391

Disruptionof CellsReleases Their Organelles and Other Contents

391

CentrifugationCan SeparateMany Typesof Organelles

392

Organelle-Specific AntibodiesAre Usefulin P r e p a r i n gH i g h l yP u r i f i e dO r g a n e l l e s

393

l s o l a t i o nC , u l tu re a , n d Differentiation 394 of MetazoanCells Flow CytometrySeparatesDifferent CellTypes

394

C u l t u r eo f A n i m a l C e l l sR e q u i r e sN u t r i e n t - R i c h M e d i a a n d S p e c i aSl o l i dS u r f a c e s

395

P r i m a r yC e l lC u l t u r e sC a n B e U s e dt o S t u d yC e l l Differentiation

396

P r i m a r yC e l lC u l t u r e sa n d C e l lS t r a i n sH a v ea F i n i t e L i f eS p a n

396

TransformedCellsCan Grow Indefinitelyin Culture

397

SomeCell LinesUndergo Differentiationin Culture

398

H y b r i dC e l l sC a l l e dH y b r i d o m a sP r o d u c eA b u n d a n t M o n o c l o n aA l ntibodies

400

HATMedium ls CommonlvUsedto lsolate HybridCells

402

organelles 407 Cnsstc ExprRturrur9.1 separating

ET R U C T U R E 1 O B I O M E M B R A NS s[

409

B i o m e mb ra n e L s:i p i dC o mp o sition 411 a n i za ti o n a n d S t r u ctu raOrg l

P h o s p h o l i p i dSsp o n t a n e o u s lFyo r mB i l a y e r s

411

B i l a y e r sF o r ma S e a l e dC o m p a r t m e n t Phospholipid S u r r o u n d i n ga n I n t e r n a lA q u e o u sS p a c e

411

M o t i f s H e l pT a r g e tP e r i p h e r a l Lipid-Binding Proteinsto the Membrane

427

ProteinsCan Be Removedfrom Membranes by Detergentsor High-SaltSolutions

427

$f,

sphingolipids, Phospholipids, SYnthesis andCholesterol: Movement and Intracellular

429

Fatty AcidsSynthesisls Mediated by Several lmportant Enzymes

430

SmallCytosolicProteinsFacilitateMovement of FattyAcids Incorporationof Fatty Acids into Membrane Lipids TakesPlaceon OrganelleMembranes

430 431

F l i p p a s eM s o v e P h o s p h o l i p i df sr o m O n e M e m b r a n e Leafletto the OppositeLeaflet

431

Cholesterolls Synthesizedby Enzymesin the Cytosol and ERMembrane

432

Cholesterola nd Phospholi pids Are Transported BetweenOrganellesby SeveralMechanisms

433

1 1 T R A N S M E M B R A NTER A N S P O R T OF IONSAND SMALLM OLECU LES437

E

overview of Membrane Transport

l lasses B i o m e m b r a n eC s o n t a i nT h r e eP r i n c i p aC of Lipids

415

M o s t L i p i d sa n d M a n y P r o t e i n sA r e L a t e r a l l yM o b i l e in Biomembranes

416

O n l y S m a l lH y d r o p h o b i cM o l e c u l e sC r o s sM e m b r a n e s 438 b y S i m p l eD i f f u s i o n

L i p i dC o m p o s i t i o nI n f l u e n c etsh e P h y s i c a l Propertiesof Membranes

418

Membrane ProteinsMediate Transportof Most Moleculesand All lons Across Biomembranes

Lipid Compositionls Different in the Exoplasmic and CytosolicLeaflets

419

s l u s t e rw i t h S p e c i f i c C h o l e s t e r oal n d S p h i n g o l i p i dC P r o t e i n si n M e m b r a n eM i c r o d o m a i n s

420

ProteinComponents Biomembranes: 421 and BasicFunctions

@

438

439

uniport Transportof Glucose and Water

441

Most TransmembraneProteinsHave M e m b r a n e - S p a n n i nagH e l i c e s

SeveralFeaturesDistinguishUniport Transportfrom S i m p l eD i f f u s i o n GLUT1UniporterTransportsGlucoseinto Most M a m m a l i a nC e l l s s F a m i l yo f S u g a r T h e H u m a nG e n o m eE n c o d e a TransportingGLUTProtetns

M u l t i p l e B S t r a n d si n P o r i n sF o r m M e m b r a n e - S p a n n i n"gB a r r e l s "

424

TransportProteinsCan Be EnrichedWithin Artificial M e m b r a n e sa n d C e l l s

CovalentlyAttached HydrocarbonChainsAnchor Some Proteinsto Membranes

424

$[|

ProteinsInteractwith Membranesin Three Different Ways

s re A l l T r a n s m e m b r a nPer o t e i n sa n d G l y c o l i p i dA A s y m m e t r i c a l lO y r i e n t e di n t h e B i l a y e r

421

OsmoticPressureCausesWater to Move Across Membranes AquaporinsIncreasethe Water Permeabilityof Cell Membranes

CONTENTS

O

44'l 442 443 443 4M 4M

xxiii

pumpsand the ATP-powered I n t r a c el l u l al ro n i cE n vi ro n me nt 447

@

Different Classes of PumpsExhibitCharacteristic Structuraland Functionalproperties ATP-Poweredlon PumpsGenerateand M a i n t a i nl o n i cG r a d i e n t sA c r o s sC e l l u l a r Membranes MuscleRelaxationDependson Ca2*ATpases That Pump Ca" from the Cytosolinto the S a r c o p l a s mR i ce t i c u i u m CalmodulinRegulates the plasmaMembrane Ca'* PumpsThat Control CytosolicCa2+ Concentrations N a - / K - A T P a s eM a i n t a i n st h e I n t r a c e l l u l aN r a* a n d K * C o n c e n t r a t i o nisn A n i m a l C e l l s V-ClassH* ATPases Maintain the Acidity of Lysosomes and Vacuoles BacterialPermeases Are ABC proteinsThat lmport a Variety of Nutrientsfrom the Environment

N o n g a te dto n C h a n n e l a s n d th e potential RestingMembrane

SelectiveMovement of lons Createsa TransmembraneElectricpotential Difference T h e M e m b r a n eP o t e n t i a li n A n i m a l C e l l sD e p e n d s Largelyon Potassiumlon MovementsThrough O p e n R e s t i n gK + C h a n n e l s

467

Na*-LinkedCa2*Antiporter ExportsCa2* from C a r d i a cM u s c l eC e l l s 447

448

449

451 452 453

454

The Approximately50 MammalianABCTransporters PlayDiverseand lmportant Rolesin Cell and O r g a nP h y s i o l o g y 455 C e r t a i nA B CP r o t e i n s" F l i p ' ,p h o s p h o l i p i d s and Other Lipid-SolubleSubstratesfrom One Membrane Leafletto the Opposite Leaflet 456

El

BacterialSymporterStructureRevealsthe Mechanismof SubstrateBinding

SeveralCotransportersRegulate CytosolicpH

468

A PutativeCation ExchangeProtein Playsa K e y R o l ei n E v o l u t i o no f H u m a nS k i n Pigmentation

469

NumerousTransportProteinsEnablePlant Vacuolesto AccumulateMetabolitesand lons

469

ft|

Transeprrnerar Transporr

Multiple TransportProteinsAre Neededto Move Glucoseand Amino AcidsAcross Epithelia

471

SimpleRehydrationTherapyDependson the OsmoticGradientCreatedby Absorption o f G l u c o s ea n d N a +

411

ParietalCellsAcidify the StomachContentsWhile M a i n t a i n i n ga N e u t r a lC y t o s o l i cp H

472

Cusslc ExprntueruT 11.1 stumbting Upon ActiveTransport

477

12 CELLULAR ENERGETICS 458

@ 458

470

FirstStepsof Glucoseand Fatty Acid CatabolismGlycolysis : and the CitricAcid €ycle

479

480

(Stagel), CytosolicEnzymes During Glycolysis ConvertGlucoseto Pyruvate

481

460

lon ChannelsContain a SelectivityFilter Formed from ConservedTransmembraneSegments

The Rate of Glycolysis ls Adjustedto Meet the Cell'sNeed for ATP

483

461

Glucosels FermentedUnder AnaerobicConditions

PatchClampsPermit Measurementof lon M o v e m e n t sT h r o u g hS i n g l eC h a n n e l s

485

463

U n d e r A e r o b i cC o n d i t i o n s ,M i t o c h o n d r i a E f fi c i e n t l yO x i d i z eP y r u v a t ea n d G e n e r a t e ATP (Stagesll-lV)

485

464

MitochondriaAre DynamicOrganelleswith Two Structurallyand FunctionallyDistinctMembranes

485

464

In Stagell, Pyruvatels Oxidizedto CO2and HighEnergyElectronsStored in ReducedCoenzymes

487

Novel lon ChannelsCan Be Characterizedby a Combinationof Oocyte Expression and P a t c hC l a m p i n g N a - E n t r yi n t o M a m m a l i a nC e l l sH a sa N e g a t i v e Changein FreeEnergy(AG)

Cotransportby symportersand Antiporters

@|

N a * - L i n k e dS y m p o r t e r lsm p o r t A m i n o A c i d sa n d G l u c o s ei n t o A n i m a l C e l l sA g a i n s tH i g h ConcentrationGradients

xxiv

.

coNTENTs

465

466

Transportersin the Inner MitochondrialMembrane H e l p M a i n t a i nA p p r o p r i a t eC y t o s o l i a c nd Matrix Concentrationsof NAD* and NADH MitochondrialOxidation of Fatty AcidsGenerates ATP PeroxisomalOxidation of Fatty AcidsGenerates No ATP

491

@ Th e

El e ctro nT ra n sp o rtch a i n a n d Generationof the Proton-Motive 493 Force

the StepwiseElectronTransportEfficientlyReleases EnergyStored in NADH and FADH2

493

ElectronTransportin Mitochondria ls Coupledto P r o t o nP u m p i n g

493

ElectronsFlow from FADH2and NADHto 02 Through Four Multiprotein Complexes

PhotoelectronTransportfrom EnergizedReactionCenterChlorophylla Producesa Charge Separation

514

I n t e r n a lA n t e n n a a n d L i g h t - H a r v e s t i n g C o m p l e x e sI n c r e a s et h e E f f i c i e n c yo f Photosynthesis

515

494

MolecularAnalysisof Photosystems

517

ReductionPotentialsof ElectronCarriersFavor ElectronFlow from NADH to 02

499

The SinglePhotosystemof PurpleBacteria Generatesa Proton-MotiveForcebut No 02

517

ExperimentsUsing PurifiedComplexesEstablished the Stoichiometryof Proton Pumping

499

The Q CycleIncreases the Numberof Protons Translocated as ElectronsFlow Through C o m p l e xl l l

LinearElectronFlow Through Both Plant Photosystems, PSlland PSl,Generatesa Proton-MotiveForce, 519 02, and NADPH

500

A n O x y g e n - E v o l v i nC g o m p l e xl s L o c a t e do n t h e L u m i n a lS u r f a c eo f t h e P S l lR e a c t i o n Center

520

CellsUse Multiple Mechanismsto ProtectAgainst Damagefrom ReactiveOxygenSpeciesDuring PhotoelectronTransPort

521

CyclicElectronFlow Through PSIGeneratesa Proton-MotiveForcebut No NADPHor 02

522

I and ll Are RelativeActivitiesof Photosystems Regulated

523

The Proton-MotiveForcein Mitochondria ls Due Largelyto a Voltage GradientAcrossthe Inner Membrane ToxicBy-productsof ElectronTransportCan D a m a g eC e l l s

[[

Harnessingthe Proton-Motive F o r c ef o r E n e rg y-R e q u i ri n g Processes

502 502

503

[[l

The Mechanismof ATPSynthesisls Shared Among Bacteria,Mitochondria,and Chloroplasts

505

ATPSynthaseComprisesTwo Multiprotein ComplexesTermedFeand F1

505

During co2 Metabolism Photosynthesis

524

RubiscoFixesCO2in the ChloroplastStroma

525

Synthesisof SucroseUsing FixedCO2ls Completed in the Cytosol

525

506

Light and RubiscoActivaseStimulateCO2 Fixation

525

ATP-ADPExchangeAcrossthe Inner Mitochondrial Membrane ls Poweredby the Proton-Motive Force

s09

Which Competeswith Photorespiration, ls Reducedin PlantsThat Fix Photosynthesis, CO2by the C4PathwaY

527

R a t eo f M i t o c h o n d r i aO l x i d a t i o nN o r m a l l yD e p e n d s on ADP Levels

510

Brown-FatMitochondria Usethe Proton-Motive Forceto GenerateHeat

510

Rotation of the Ft 1 Subunit,Driven by Proton Movement Through F6,Powers ATPSynthesis

@

1 3 M O V I N GP R O T E I NISN T O A N D O R G A N E L L E Ss33 MEMBRANES

Ph o t o s yn th e siasn d L i g h t-A b sor bing Pigments

ThylakoidMembranesin ChloroplastsAre the Sites in Plants of Photosynthesis

511 511

Occur Three of the Four Stagesin Photosynthesis O n l y D u r i n gl l l u m i n a t i o n

511

EachPhoton of Light Hasa DefinedAmount of Energy

513

Comprisea ReactionCenterand Photosystems Complexes AssociatedLight-Harvesting

514

$[

of secretoryProteins Translocation 535 Acr ossthe ERMembr ane

A HydrophobicN-TerminalSignalSequenceTargets NascentSecretoryProteinsto the ER

536

CotranslationalTranslocationls lnitiated by Two Proteins GTP-Hydrolyzing

537

Passageof Growing PolypeptidesThrough the Transloconls Driven by EnergyReleasedDuring Translation

539

CONTENTS

.

XXV

ATPHydrolysisPowersPost-translational Translocationof SomeSecretoryProteinsin yeast

Insertionof proteinsinto the E RM e mb ra n e

$[

540

542

543 InternalStop-Transfer and Signal-AnchorSequences proteins DetermineTopologyof Single-Pass 544 M u l t i p a s sP r o t e i n sH a v eM u l t i p l e I n t e r n a l TopogenicSequences 546 A P h o s p h o l i p iA d n c h o rT e t h e r sS o m eC e l l - S u r f a c e Proteinsto the Membrane 547 The Topologyof a Membrane ProteinOften Can Be Deducedfrom lts Sequence 547

A Preformed/V-LinkedOligosaccharide ls Added to M a n y P r o t e i n si n t h e R o u g hE R O l i g o s a c c h a r i dSei d eC h a i n sM a y p r o m o t eF o l d i n g and Stabilityof Glycoproteins D i s u l f i d eB o n d sA r e F o r m e da n d R e a r r a n g e db y P r o t e i n si n t h e E RL u m e n C h a p e r o n eas n d O t h e r E Rp r o t e i n sF a c i l i t a t eF o l d i n g and Assemblyof Proteins lmproperlyFoldedProteinsin the ERlnduce Expression of Protein-FoldingCatalysts U n a s s e m b l eodr M i s f o l d e dP r o t e i n si n t h e E RA r e Often Transportedto the Cytosolfor Degradation

550

ss2

coNTENTs

569

Largeand Small MoleculesEnter and Leavethe Nucleusvia NuclearPoreComplexes

570

lmportinsTransportProteinsContainingNuclearL o c a l i z a t i oS n i g n a l si n t o t h e N u c l e u s

571

ExportinsTransportProteinsContainingNuclear-Export S i g n a l so u t o f t h e N u c l e u s 573 573

1 4 V E S I C U L ATRR A F F I CS, E C R E T I O N , AND ENDOCYTOSIS 579 Techniques for Studyingthe SecretoryPathway

s80

Transportof a protein Through the Secretory pathway Can Be Assayedin tiving Cells

5g2

YeastMutants Define Major Stagesand Many Componentsin VesicularTransport

584

Cell-FreeTransportAssaysAllow Dissectionof I n d i v i d u aS l t e p si n V e s i c u l aTr r a n s p o r t

585

![ ss2 555

556

and Chloroplasts

.

Transportinto and out of the Nucleus

s[

552

A m p h i p a t h i cN - T e r m i n aSl i g n a lS e q u e n c eD sirect Proteinsto the MitochondrialMatrix 558 M i t o c h o n d r i aP l r o t e i nl m p o r t R e q u i r e sO u t e r - M e m b r a n e R e c e p t o ras n d T r a n s l o c o ni sn B o t h M e m b r a n e s 559 Studieswith ChimericProteinsDemonstratelmportant Featuresof Mitochondriallmport 550 T h r e eE n e r g yI n p u t sA r e N e e d e dt o l m p o r t P r o t e i n s into Mitochondria 501 Multiple Signalsand PathwaysTarget Proteinsto SubmitochondrialCompartments 561 T a r g e t i n go f C h l o r o p l a sSt t r o m a lP r o t e i n sl s S i m i l a rt o lmport of MitochondrialMatrix Proteins 565 ProteinsAre Targetedto Thylakoidsby Mechanisms Relatedto TranslocationAcrossthe Bacterial I n n e rM e m b r a n e 565 xxvi

s68

549

Sortingof Proteinsto Mitochondria 557

E!|

P e r o x i s o m aM l e m b r a n ea n d M a t r i x P r o t e i n sA r e Incorporated by Different Pathways

Most mRNAsAre Exportedfrom the Nucleusby a R a n - l n d e p e n d e nMt e c h a n i s m

Pr o t e i nMo d i fi ca ti o n s, F o l d i n g, and Quality Control in the ER

Sortingof Peroxisomal Proteins sG7

CytosolicReceptorTargetsProteinswith an SKL Sequenceat the C-Terminusinto the Peroxisomal Matrix

SeveralTopologicalClasses of Integral Membrane ProteinsAre Synthesizedon the ER

s[

s[

@

M olecularMechanisms of Vesicular Traffic

Assembryof a protein coat DrivesVesicle F o r m a t i o na n d S e l e c t i o no f C a r g o Molecules A ConservedSet of GTPaseSwitch proteinsControls Assemblyof Different VesicleCoats T a r g e t i n gS e q u e n c e os n C a r g o p r o t e i n sM a k e S p e c i f i cM o l e c u l a rC o n t a c t sw i t h C o a t Proteins

585

5g6 5g7

588

Rab GTPases Control Dockingof Vesicleson Target Membranes PairedSetsof SNAREproteinsMediate Fusionof Vesicleswith Target Membranes

591

Dissociationof SNAREComplexesAfter Membrane F u s i o nl s D r i v e nb y A T PH y d r o l y s i s

5g1

589

@

EarlyStagesof the Secretory Pathway

592

COPIIVesiclesMediate Transportfrom the ER to the Golgi COPIVesiclesMediate RetrogradeTransportwithin t h e G o l g ia n d f r o m t h e G o l g i t o t h e E R

ss4

AnterogradeTransportThrough the Golgi Occurs by CisternalMaturation

595

@

Later stages of the secretory Pathway

597

VesiclesCoatedwith Clathrinand/or Adapter Proteins 598 Mediate SeveralTransportSteps

l: SIGNAL 1 5 C E L LS I G N A L I N G AND SHORT- T ER M TRANSDUCTION 623 RESPONSES CELLULAR [[

Signalto cellular FromExtracellular 625 Response

Signaling S i g n a l i n gC e l l sP r o d u c ea n d R e l e a s e Molecules

625

SignalingMoleculesCan Act Locallyor at a Distance

625

B i n d i n go f S i g n a l i n gM o l e c u l e sA c t i v a t e s Receptorson Target Cells

626

D y n a m i nl s R e q u i r e df o r P i n c h i n gO f f o f C l a t h r i n Vesicles

599

$[

Mannose6-PhosphateResiduesTargetSoluble Proteinsto Lysosomes

600

ReceptorProteinsBind LigandsSpecifically

627

RevealedKey Study of LysosomalStorageDiseases Componentsof the LysosomalSorting Pathway

602

The DissociationConstantls a Measureof the Affinity of a Receptorfor lts Ligand

628

ProteinAggregation in the trans-GolgiMay Function in Sorting Proteinsto RegulatedSecretoryVesicles 602

BindingAssaysAre Usedto Detect Receptorsand DetermineTheir Affinities for Ligands

628

Some ProteinsUndergo ProteolyticProcessing After Leavingthe trans-Golgi

603

SeveralPathwaysSort Membrane Proteinsto the Apical or BasolateralRegionof PolarizedCells

to a SignalingMolecule MaximalCellularResponse UsuallyDoesNot RequireActivationof All Receptors

629

604

Sensitivityof a Cellto ExternalSignalsls Determined by the Number of SurfaceReceptorsand Their 631 Affinity for Ligand

Receptor-Mediated Endocytosis 606

631 ReceptorsCan Be Purifiedby Affinity Techniques ReceptorsAre FrequentlyExpressedfrom Cloned Genes 6 3 1

!f|

CellsTake Up Lipidsfrom the Blood in the Form of Large,Well-Defined LipoproteinComplexes

608

The Acidic pH of Late EndosomesCausesMost Receptor-Ligand Complexesto Dissociate

610

[!|

DirectingMembraneProteins and CytosolicMaterialsto the Lysosome

MultivesicularEndosomesSegregateMembrane ProteinsDestinedfor the Lysosomal Membranefrom ProteinsDestinedfor LysosomalDegradation

627

606

Receptorsfor Low-DensityLipoproteinand Other LigandsContain Sorting SignalsThat Target Them for Endocytosis

The EndocyticPathwayDeliverslron to Cellswithout Complex Dissociationof the Receptor-Transferrin in Endosomes

studying cell-Surface Receptors

611

Highly ConservedComPonents Signal-Transduction of Intracellular 632 Pathways GTP-BindingProteinsAre FrequentlyUsedAs On/Off Switches are Employedin Protein Kinasesand Phosphatases Virtually All SignalingPathways Carryand Amplify Signalsfrom SecondMessengers Many Receptors

612

612

RetrovirusesBud from the PlasmaMembrane by a Process Endosomes 614 Similarto Formationof Multivesicular

14.1 Following a Protein Cnsstc ExprRturruT 621 out of thecell

s!|

633 634 634

of G ProteinGeneralElements 535 SYstems RecePtor Coupled

G Protein-CoupledReceptorsAre a Largeand Diverse 635 Familywith a Common Structureand Function G Protein-CoupledReceptorsActivate Exchangeof G T Pf o r G D Po n t h e a S u b u n i to f a T r i m e r i cG 637 Protein Different G ProteinsAre Activated by Different GPCRs 539 and ln Turn RegulateDifferent EffectorProteins

CONTENTS

.

xxvii

G Protein-Coupled Receptors T h a t R eg u l a tel o n C h a n n e l s

640

AcetylcholineReceptorsin the Heart Muscle Activate a G ProteinThat OpensK+ Channels

641

Light ActivatesG..-CoupledRhodopsins

641

Activationof RhodopsinInducesClosingof cGMP-GatedCation Channels

642

[f|

Rod CellsAdapt to Varying Levelsof Ambient Light Becauseof Opsin Phosphorylationand Binding of Arrestin

644

G Protein-GoupledReceptorsThat Activateor Inhibit Adenylyl Cyclase 646

$!|

Adenylyl Cyclasels Stimulatedand Inhibited by Different Receptor-Ligand Complexes

646

StructuralStudiesEstablishedHow G,,.GTpBinds to and ActivatesAdenylyl Cyclase

646

cAMP ActivatesProtein KinaseA by Releasing CatalyticSubunits

547

GlycogenMetabolismls Regulatedby Hormone-lnducedActivation of Protein KinaseA

648

cAMP-MediatedActivation of Protein KinaseA ProducesDiverseResponses in Different Cell Types

649

S i g n a lA m p l i f i c a t i o nC o m m o n l yO c c u r si n M a n y SignalingPathways

550

S e v e r aM l e c h a n i s mD s o w n - R e g u l a tS e ignaling from G Protein-CoupledReceptors

651

Anchoring ProteinsLocalizeEffectsof cAMp to SpecificRegionsof the Cell

ActivatePhospholipase C

PhosphorylatedDerivativesof InositolAre lmportant SecondMessengers C a l c i u ml o n R e l e a s e from the Endoplasmic Reticulumis Triggeredby lp3 The Ca2*/Calmodulin ComplexMediatesMany CellularResponses to ExternalSignals Diacylglycerol(DAG)Activatesprotein KinaseC, Which RegulatesMany Other proteins Signal-lnducedRelaxationof VascularSmooth Musclels Mediated by cGMp-Activated Protein KinaseG

Clnsstc ExprnlveruT 15.1 TheInfancy of signal Transduction-GTPStimulationof cAMP Synthesis 663

15 CELL-SIGNALIN l lG : SIGNALING PATHWAYSTHAT C O N T R O L GENE ACTIVITY 66s

sl|

IntegratingResponses of cells t o En v i ro n me n taInl fl u e n ce s

'

CONTENTS

668 668

RadioactiveTaggingWas Usedto ldentify TGFp Receptors

669

ActivatedTGFBReceptorsPhosphorylateSmad TranscriptionFactors

670

NegativeFeedbackLoopsRegulateTGFB/Smad Signaling

671

Lossof TGFBSignalingPlaysa Key Role in Cancer

671

sf|

cytokineReceptors andthe JAK/STAT Pathway

672 672

CytokineReceptorsHaveSimilarStructuresand Activate SimilarSignalingPathways

673

JAK KinasesActivate STATTranscriptionFactors

674

654

ComplementationGeneticsRevealedThat JAK and STATProteinsTransduceCytokine Signals

677

654

Signalingfrom CytokineReceptorsls Regulated by NegativeSignals

678

655

Mutant ErythropoietinReceptorThat Cannot Be TurnedOff Leadsto IncreasedNumbersof Erythrocytes

679

553

6s6

656

657

Integrationof Multiple SecondMessengersRegulates Glycogenolysis 557

XXVIII

Activation of Smads

A TGFBSignalingMoleculels Formedby Cleavage of an InactivePrecursor

ReceprorTyrosrneKrnases

679

L i g a n dB i n d i n gL e a d st o P h o s p h o r y l a t i oann d Activation of IntrinsicKinasein RTKs

680

Overexpression of HER2,a Receptor TyrosineKinase,Occursin Some Breast Cancers

680

s[ $f|

TGFFReceptors and the Direct

CytokinesInfluenceDevelopmentof Many Cell Types

G Protein-coupled Receptors That

sfl

I n s u l i na n d G l u c a g o nW o r k T o g e t h e rt o M a i n t a i na StableBlood GlucoseLevel

HedgehogSignalingRelievesRepressionof Target Genes

D o m a i n sA r e l m p o r t a n tf o r B i n d i n gS i g n a l Conserved TransductionProteinsto ActivatedReceptors Down-regulationof RTKSignalingOccursby Endocytosis and LysosomalDegradation

683

@

700

PathwaysThat InvolveSignal - l nduc ed 703 ProteinCleavage

o f R a sa n d MA P K i n ase @ Act i v a t i o n 584 Pathways

Degradationof an Inhibitor ProteinActivatesthe NF-rBTranscriptionFactors

703

Ras,a GTPaseSwitch Protein,CyclesBetweenActive and InactiveStates

68s

Ligand-ActivatedNotch ls CleavedTwice,Releasing a TranscriptionFactor

705

ReceptorTyrosineKinasesAre Linkedto Rasby Adapter Proteins

685

GeneticStudiesin Drosophilaldentified Key Proteinsin the Ras/MAP Signal-Transducing KinasePathway

685

Binding of SosProteinto InactiveRasCausesa ConformationalChangeThat ActivatesRas

587

SignalsPassfrom Activated Rasto a Cascadeof Protein Kinases

688

MAP KinaseRegulatesthe Activity of Many Transcription 690 Genes FactorsControlling Early-Response G Protein-CoupledReceptorsTransmitSignalsto MAP Kinasein YeastMating Pathways

691

ScaffoldProteinsSeparateMultiple MAP Kinase Pathwaysin EukaryoticCells

692

The Ras/MAPKinasePathwayCan InduceDiverse C e l l u l a rR e s p o n s e s

693

sf|

Phosphoinositides as signal Transducers

Phospholipase C" ls Activatedby SomeRTKsand CytokineReceptors

694

6e4

Recruitmentof Pl-3 Kinaseto Hormone-Stimulated ReceptorsLeadsto Synthesisof Phosphorylated Phosphatidyl inositols in the Plasma Accumulationof Pl 3-Phosphates Membrane Leadsto Activation of SeveralKinases 695 Activated Protein KinaseB InducesMany CellularResponses

696

The Pl-3KinasePathwayls NegativelyRegulated by PTENPhosphatase

s!|

698

Arrestin ActivatesSeveralKinaseCascades 698 GPCR-Bound Wnt SignalsTrigger Releaseof a Transcription Factorfrom CytosolicProteinComplex

RegulatedIntramembraneProteolysisof SREBP a TranscriptionFactorThat Acts to Releases Maintain Phospholipidand CholesterolLevels

1 7 C E L LO R G A N I Z A T I OANN D 713 M O V E M E N Tl : M I C R O F I L A M E N T S

@

and Actin Microfilaments Structures

715

Actin ls Ancient,Abundant, and Highly Conserved G-ActinMonomersAssembleinto Long, Helical F-ActinPolymers

717

F-ActinHasStructuraland FunctionalPolarity

718

@

Dynamicsof Actin Filaments

717

718

Actin Polymerizationin Vitro Proceedsin Three Steps 7 1 9 Actin FilamentsGrow Fasterat (+) EndsThan at 720 (-) Ends Actin FilamentTreadmillingls Acceleratedby P r o f i l i na n d C o f i l i n Providesa Reservoirof Actin for Thymosin-B4 Polymerization CappingProteinsBlockAssemblyand Disassembly a t A c t i n F i l a m e n tE n d s

Activationof GeneTranscription Cell-Surface by Seven-spanning 697 Receptors

CREBLinkscAMP and Protein KinaseA to Activation of GeneTranscription

CatalyzeCleavageof Many Matrix Metalloproteases 706 S i g n a l i n gP r o t e i n sf r o m t h e C e l lS u r f a c e InappropriateCleavageof Amyloid PrecursorProtein Can Leadto Alzheimer'sDisease

@

of Actin Filament Mechanisms AssemblY

F o r m i n sA s s e m b l eU n b r a n c h e dF i l a m e n t s The Arp2/3 ComplexNucleatesBranchedFilament Assembly lntracellularMovementsCan Be Poweredby Actin Polymerization

CONTENTS

721 722 722

723 723 724 726 xxix

ToxinsThat Perturbthe Pool of Actin Monomers Are Usefulfor StudyingActin Dynamics

726

ChemotacticGradientsInduceAltered Phosphoinositide LevelsBetweenthe Front and Backof a Cell 750

Cnsstc ExpenlvlrruT 17.1 Looking at Muscle

o r g a n i z a ti o no f A cti n -B a secellular d Structures 728

@

C r o s s - L i n k i nPgr o t e i n sO r g a n i z eA c t i n F i l a m e n t si n t o Bundlesor Networks

728

Adaptor ProteinsLink Actin Filamentsto Membranes

728

Myosins: Actin-Based Motor Proteins

fifl

Contraction

755

1 8 C E L LO R G A N I Z A T I OANN D M O V E M E N Tl l : M I C R O T U B U L E S AND INTERMEDIATE FILAM EN T S 757 [[

731

Micr otubuleStr uctur eand Or ganization

758

732

MicrotubuleWalls Are PolarizedStructuresBuilt f r o m a B - T u b u l i nD i m e r s

758

M y o s i n sM a k e U p a L a r g eF a m i l yo f MechanochemicalMotor proteins

733

MicrotubulesAre Assembledfrom MTOCsto GenerateDiverseOrganizations

760

C o n f o r m a t i o n aCl h a n g e si n t h e M y o s i nH e a d CoupleATPHydrolysisto Movement

736

Myosin HeadsTake DiscreteStepsAlong Actin Filaments

736

MyosinV Walks Hand Over Hand Down an Actin Filament

MicrotubulesAre DynamicStructuresDue to Kinetic Differencesat Their Ends

763

737

I n d i v i d u aM l i c r o t u b u l e sE x h i b i tD y n a m i cI n s t a bIi t y

763

LocaIized A s s e m b l ya n d " S e a r c h - a n d - C a p t u r e " H e l p O r g a n i z eM i c r o t u b u l e s

766

DrugsAffecting Tubulin PolymerizationAre Useful Experimentallyand to Treat Diseases

766

M y o s i n sH a v eH e a d ,N e c k ,a n d T a i l D o m a i n sw i t h DistinctFunctions

Myosin-poweredMovements

@

M y o s i nT h i c kF i l a m e n t sa n d A c t i n T h i n F i l a m e n t s i n S k e l e t aM l u s c l eS l i d eP a s tO n e A n o t h e r D u r i n gC o n t r a c t i o n

738

ftf|

738

[f!

S k e l e t aM l u s c l el s S t r u c t u r e db y S t a b i l i z i n ga n d ScaffoldingProteins

740

Contractionof SkeletalMusclels Regulatedby Ca2* a n d A c t i n - B i n d i n gP r o t e i n s

740

Actin and Myosin ll Form ContractileBundlesin N o n m u s c l eC e l l s

741 Myosin-DependentMechanismsRegulate C o n t r a c t i o ni n S m o o t hM u s c l ea n d N o n m u s c l eC e l l s 742 Myosin-V-Bound VesiclesAre CarriedAlong Actin Filaments 743

Microtubule Dynamics

762

Regulationof MicrotubuleStructure and Dynam ics 767

MicrotubulesAre Stabilizedby Side-and End-Binding Proteins

767

M i c r o t u b u l e sA r e D i s a s s e m b l ebdy E n d B i n d i n g and SeveringProteins

768

@

Kinesinsand Dyneins:Micr otubul eBasedMotor Proteins 769

Organellesin AxonsAre TransportedAlong Microtubulesin Both Directions

769

Kinesin-1PowersAnterogradeTransportof VesiclesDown Axons Toward the (+) End of Microtubules

770

745

KinesinsForm a LargeProtein Familywith Diverse Functions

771

747

Kinesin-l ls a Highly Processive Motor

772

C e l lM i g r a t i o nI n v o l v e st h e C o o r d i n a t eR e g u l a t i o n of Cdc42,Rac,and Rho

748

Dynein Motors TransportOrganellesTowardthe (-) E n do f M i c r o t u b u l e s

774

Migrating CellsAre Steeredby Chemotactic Molecules

750

Kinesinsand DyneinsCooperatein the Transport o f O r g a n e l l eT s hroughout he Cell

715

c e l l M i g r a t i o ns: i g n a l i n ga n d Chemotaxis

@

C e l lM i g r a t i o nC o o r d i n a t e F s o r c eG e n e r a t i o nw i t h C e l lA d h e s i o na n d M e m b r a n eR e c y c l i n g The SmallGTP-BindingProteinsCdc42,Rac,and Rho Control Actin Organization

XXX

.

CONTENTS

745

fifl

Ciliaand Flagella: Microtubule777 BasedSurfaceStructures

E u k a r y o t i cC i l i aa n d F l a g e l l aC o n t a i nL o n g D o u b l e t MicrotubulesBridged by Dynein Motors

777

r e a t i n gA r e P r o d u c e db y C i l i a r ya n d F l a g e l l a B C o n t r o l l e dS l i d i n go f O u t e r D o u b l e t Microtubules

778

IntraflagellarTransportMoves Material Up and D o w n C i l i aa n d F l a g e l l a

779

Defectsin IntraflagellarTransportCauseDisease by Affecting SensoryPrimaryCilia

780

Microfilamentsand MicrotubulesCooperateto TransportMelanosomes Cdc42Coordi nates M icrotubules and M icrofi laments D u r i n g C e l lM i g r a t i o n

1 9 I N T E G R A T I NCGE L L SI N T O TISSUES

$[

797

801

cell- celland cell- Matr ixAdhes i on: 803 An Overview

C e l l - A d h e s i oM n o l e c u l e sB i n dt o O n e A n o t h e r a n d t o I n t r a c e l l u l aPr r o t e i n s

803

782

805

C e n t r o s o m eD s u p l i c a t eE a r l yi n t h e C e l lC y c l ei n Preparationfor Mitosis

The ExtracellularMatrix Participatesin Adhesion, S i g n a l i n ga, n d O t h e r F u n c t i o n s

783

807

of The Mitotic SpindleContainsThree Classes Microtubules

The Evolutionof MultifacetedAdhesionMolecules Enabledthe Evolutionof DiverseAnimal Tissues

784

M i c r o t u b u l eD y n a m i c sI n c r e a s eDs r a m a t i c a l l y in Mitosis

784

$!|

Mitosis

MitosisCan Be Dividedinto Six Phases

781

l u r i n gM i t o s i s M i c r o t u b u l e sT r e a d m i l D

785

The KinetochoreCapturesand HelpsTransport Chromosomes

786

DuplicatedChromosomesAre Aligned by Motors a n d T r e a d m i l l i n gM i c r o t u b u l e s

788

to Polesby AnaphaseA MovesChromosomes Microtubule Shortening

789

AnaphaseB SeparatesPolesby the Combined A c t i o n o f K i n e s i n sa n d D y n e i n

789

s o n t r i b u t et o S D i n d l e A d d i t i o n a lM e c h a n i s mC Formation

789

CytokinesisSplitsthe DuplicatedCell in Two

789

e h e i r M i c r o t u b u l e sa n d P l a n tC e l l sR e o r g a n i z T B u i l da N e w C e l l W a l li n M i t o s i s

790

$fl

IntermediateFilaments

791

@

Junctio ns cell- celland celI- ECM and TheirAdhesionMolecules 808

, nd t p i c a l ,L a t e r a l a E p i t h e l i aC l e l l sH a v eD i s t i n c A BasalSurfaces

808

ThreeTypesof JunctionsMediate Many Cell-Cell and Cell-ECMInteractions

809

l l d h e s i o n si n A d h e r e n s C a d h e r i n sM e d i a t eC e l l - C e A J u n c t i o n sa n d D e s m o s o m e s

810

Tight JunctionsSealOff Body Cavitiesand Restrict D i f f u s i o no f M e m b r a n eC o m p o n e n t s

814

Adhesionsin EpithelialCells 816 IntegrinsMediateCelI-ECM s llow Small G a p J u n c t i o n sC o m p o s e do f C o n n e x i n A Moleculesto PassDirectlyBetweenAdjacent Cells 817

M atr ix l: The Extr acellular The BasalLamina

820

l a m i n aP r o v i d e sa F o u n d a t i o nf o r The BasaL Assemblyof Cellsinto Tissues

820 821

IntermediateFilamentsAre Assembledfrom S u b u n i tD i m e r s

792

M a t r i x P r o t e i n ,H e l p s L a m i n i n ,a M u l t i a d h e s i v e Cross-linkComponentsof the BasalLamina

Intermediate FilamentsProteins Are Expressed Manner in a Tissue-Specific

792

Type lV Collagenls a Major Structural Sheet-Forming 821 l amina C o m p o n e n to f t h e B a s a L

I n t e r m e d i a t eF i l a m e n t sA r e D y n a m i c

795

Defectsin Laminsand KeratinsCauseManv Diseases 795

[[

coordinationand cooperation between CytoskeletalElements 796

Proteins lntermediateFilament-Associated C o n t r i b u t et o C e l l u l a rO r g a n i z a t i o n

796

Components Perlecan,a Proteoglycan,Cross-links Receptors of the BasalLaminaand Cell-Surface

@The

Mra t r i xl l : Extracellula Connectiveand Other Tissues

s r e t h e M a j o r F i b r o u sP r o t e i n s F i b r i l l a rC o l l a g e n A in the ECMof ConnectiveTissues

CONTENTS

824

825 825

XXXI

F i b r i l l a rC o l l a g e nl s S e c r e t e da n d A s s e m b l e di n t o FibrilsOutsideof the Cell

926

ConnectionsBetweenthe ECMand Cytoskeleton Are Defectivein MuscularDystrophy

835

T y p eI a n d l l C o l l a g e n A s ssociate with Nonfibrillar Collagensto Form DiverseStructures

826

l g C A M sM e d i a t eC e l l - C e l l A d h e s i oi n N e u r o n a l and Other Tissues

836

LeukocyteMovement into Tissuesls Orchestrated by a Precisely Timed Sequenceof Adhesive Interactions

837

Proteoglycans and Their ConstituentGAGsPlay DiverseRolesin the ECM

827

HyaluronanResists Compression, Facilitates Cell Migration, and GivesCartilagelts Gel-likeProperties 829 FibronectinsInterconnectCellsand Matrix, Influencing Cell Shape,Differentiation,and Movement 830

AdhesiveInteractionsin Motile a n d N o n mo ti l eC e l l s

[[

I n t e g r i n sR e l a yS i g n a l sB e t w e e nC e l l sa n d T h e i r T h r e e - D i m e n s i o nE an l vironment

p[

PtantTissues

839

T h e P l a n tC e l lW a l l l s a L a m i n a t eo f C e l l u l o s eF i b r i l s in a Matrix of Glycoproteins

833 833

Regulationof Integrin-MediatedAdhesionand S i g n a l i n gC o n t r o l sC e l l M o v e m e n t

Looseningof the Cell Wall PermitsPlant Cell Growth

840

Plasmodesmata DirectlyConnectthe Cytosolsof A d j a c e n tC e l l si n H i g h e rP l a n t s Only a Few AdhesiveMoleculesHave Been ldentified in Plants

841

PartlV CellGrowth and Development 20 R E G U L AT IN T GH E E U K A R Y OT IC C E L LC Y C L E 847 Overviewof the CellCycle and lts Control

E[

849

The Cell Cyclels an Ordered Seriesof Events L e a d i n gt o C e l lR e p l i c a t i o n

849

RegulatedProtein Phosphorylationand DegradationControl Passage Through the Cell Cycle

849

DiverseExperimentalSystemsHave Been Usedto ldentify and lsolateCell-Cycle Control proteins

851

MPF Activity

853

Maturation-PromotingFactor(MpF)Stimulates Meiotic Maturation in Oocytesand Mitosis i n S o m a t i cC e l l s

854

Mitotic CyclinWas Firstldentified in EarlvSea U r c h i nE m b r y o s

856

CyclinB Levelsand KinaseActivity of MitosisPromoting Factor(MPF)ChangeTogetherin CyclingXenopusEgg Extracts Anaphase-Promoting Complex(APC/C)Controls Degradationof Mitotic Cyclinsand Exit from Mitosis

.

CONTENTS

8s6

8s8

Cyclin-Dependent KinaseRegulation Dur ingMitosis 8s9

MPFComponentsAre ConservedBetween Lower and Higher Eukaryotes

860

Phosphorylationof the CDKSubunit Regulatesthe KinaseActivity of MPF

851

ConformationalChangesInducedby Cyclin Binding and PhosphorylationIncrease MPFActivity

862

@

Controlof Mitosisby Cyclinsand

@

![fl

MolecularMechanisms for Regulating Mitotic Events

864

Phosphorylationof NuclearLaminsand Other ProteinsPromotesEarlyMitotic Events

864

U n l i n k i n go f S i s t e rC h r o m a t i d sI n i t i a t e sA n a p h a s e

867

ChromosomeDecondensationand Reassembly of the NuclearEnvelopeDependon Dephosphorylation of MPFSubstrates

870

!!f|

Cyclin-cDK and Ubiquitin-protein Ligase Control of S phase

872

A Cyclin-Dependent Kinase(CDK)ls Criticalfor S-PhaseEntry in S. cerevisiae

872

Three G1CyclinsAssociatewith 5. cerevrsrae CDK to Form S-Phase-Promoting Factors

874

Degradationof the S-Phase Inhibitor TriggersDNA Replication Multiple CyclinsRegulatethe KinaseActivity of 5. cerevisiaeCDK During Different Cell-CyclePhases 877 Replication a t E a c hO r i g i n l s I n i t i a t e dO n l y O n c e During the Cell Cycle

!fil

877 |ffirhe

cell-cyclecontrolin Mammalian Cells

2 1 C E L LB I R T H L, I N E A G EA, N D DEATH

879

90s

Birthof cells:Stemcells, 905

N i c h e s ,a n d L i n e a g e

Stem CellsGive Riseto Both Stem Cellsand DifferentiatingCells

906

RestrictedDuring Cell FatesAre Progressively Development

907

The CompleteCell Lineageof C. elegansls Known

908

M a m m a l i a nR e s t r i c t i o n P o i n t l s A n a l o g o u st o STARTin Yeast Cells

880

Multiple CDKsand CyclinsRegulatePassage of M a m m a l i a nC e l l sT h r o u g ht h e C e l lC y c l e

881

HeterochronicMutants ProvideCluesAbout Control of Cell Lineage

909

RegulatedExpression of Two Classes of Genes R e t u r n sG eM a m m a l i a nC e l l st o t h e C e l lC y c l e

881

Cultured EmbryonicStem CellsCan Differentiateinto V a r i o u sC e l lT y p e s

911

Passage Through the RestrictionPoint Dependson Phosphorylationof the Tumor-Suppressor Rb Protein 882

Adult Stem Cellsfor Different Animal TissuesOccupy 912 S u s t a i n i n gN i c h e s

C y c l i nA l s R e q u i r e df o r D N A S y n t h e s iasn d C D K 1 for Entry into Mitosis

883

MeristemsAre Nichesfor Stem Cellsin Postnatal Plants

Two Typesof Cyclin-CDKInhibitorsContributeto Cell-Cycle Control in Mammals

883

@

!![

in cell-Cycle Checkpoints Regulation

The Presenceof UnreplicatedDNA PreventsEntry into Mitosis lmproper Assemblyof the Mitotic SpindlePrevents the Initiation of Anaphase ProperSegregationof Daughter Chromosomesls Monitored by the Mitotic Exit Network

884 888

92O

cell-Typespecificationin Yeast

921

Mati ng-TypeTranscription FactorsSpecify CellTypes

922

MCMl and a1-MCM1ComplexesActivate Gene Transcription

923

a2-MCMI and cr2-a1ComplexesRepressTranscription 923 888

PheromonesInduceMating of ct and a Cellsto G e n e r a t ea T h i r d C e l l T Y P e

923

889

Arrest of Cellswith DamagedDNA Depends Cell-Cycle 891 on Tumor Suppressors

!!|

and Differentiation Specification 924 of Muscle

EmbryonicSomitesGive Riseto Myoblasts

925

MyogenicGenesWere Firstldentified in Studieswith Cultured Fibroblasts

925

Two Classesof Regulatory FactorsAct in Concert to Guide Productionof MuscleCells

926

CohesinSubunit Recombinationand a Meiosis-Specific for the SpecializedChromosome Are Necessary 895 Segregationin Meiosis|

Differentiationof Myoblastsls Under Positiveand NegativeControl

927

Cell-CellSignalsAre Crucialfor Determinationand Migration of Myoblasts

928

SpecialPropertiesof Rec8Regulatelts Cleavagein M e i o s i sI a n d l l

896

bHLH RegulatoryProteinsFunctionin Creationof Other Tissues

929

The Monopolin ComplexCo-orientsSister Kinetochoresin Meiosis|

898

Tensionon SpindleMicrotubulesContributesto ProperSpindleAttachment

898

!![

Meiosis:A specialTypeof cell Division

Key FeaturesDistinguishMeiosisfrom Mitosis

892 892

Protein of G1Cyclinsand a Meiosis-Specific Repression 895 KinasePromote Premeiotic5 Phase

Emerging 20.1 cellBiology Cnsstc ExpenlueruT from the Sea:The Discoveryof Cyclins

903

@

Regulationof Asymmetric CellDivision

930

YeastMating-TypeSwitchingDependsupon AsymmetricCell Division

CONTENTS

930 '

xxxiii

ProteinsThat RegulateAsymmetryAre Localizedat OppositeEndsof Dividing Neuroblastsin Drosophila 9 3 1

cell Deathand tts Regulation

!@

936

ProgrammedCell Death OccursThrough Apoptosis Neurotrophinspromote Survivalof Neurons

937

A Cascadeof CaspaseProteinsFunctionsin One Apoptotic Pathway

938

Pro-ApoptoticRegulatorsPermit CaspaseActivation in the Absenceof TrophicFactors

941

SomeTrophicFactorsInduceInactivationof a Pro-ApoptoticRegulator

942

Tumor NecrosisFactorand RelatedDeath Signals PromoteCell Murder by Activating Caspases

943

937

2 2 T H EM O L E C U L ACRE L LB I O L O G Y O FD E V E L O P M E N T 949

Controlof Body Segmentation: Themesand Variationsin Insects and Vertebrates 969 Early Drosophila Development ls an Exercisein Speed 970 TranscriptionalControl Specifiesthe Embryo's Anterior and Posterior

971

TranslationInhibitorsReinforceAnterior-Posterior Patterning

973

InsectSegmentationls Controlledby a Cascade of Transcription Factors

974

VertebrateSegmentationls Controlledby Cyclical Expression of RegulatoryGenes

977

DifferencesBetweenSegmentsAre Controlledby Hox Genes

978

Hox-GeneExpressionls Maintained by a Variety of Mechanisms

982

Flower DevelopmentRequiresSpatiallyRegulated Productionof TranscriptionFactors

983

!![ H i g h l i g h tso f D e ve l o p me n t

@

DevelopmentProgresses from Egg and Sperm to an EarlyEmbryo

As the EmbryoDevelops,Cell LayersBecomeTissues and Organs 951 GenesThat RegulateDevelopmentAre at the Heart of Evolution

Neural Development

985

N e u r u l a t i o nB e g i n sF o r m a t i o no f t h e B r a i na n d SpinalCord

986

950 9s0

952

cell-TypeSpecification in Early

SignalGradientsand TranscriptionFactorsSpecifyCell Typesin the NeuralTube and Somites 987 Most Neuronsin the BrainArise in the Innermost NeuralTube and Migrate Outward

988

LateralInhibition Mediated by Notch Signaling CausesEarlyNeural Cellsto BecomeDifferent

988

Gametogenesis and Fertilization g53

@

Growth and Patterningof Limbs 990

G e r m - l i n eC e l l sA r e A l l T h a t W e I n h e r i t

953

!![

FertilizationUnifiesthe Genome

955

Hox GenesDeterminethe Right Placesfor Limbsto Grow

990

958

Limb DevelopmentDependson Integration o f M u l t i p l e E x t r a c e l l u l aSri g n a lG r a d i e n t s

991

958

Hox GenesAlso Control Fine Patterning of Limb Structures

992

So Fa[ So Good

994

Genomiclmprinting ControlsGene Activation Accordingto Maternal or PaternalChromosome Origin Too Much of a Good Thing: The X Chromosomels Regulatedby DosageCompensation

cell Diversityand patterning

!f,

in EarlyVertebrateEmbryos

CleavageLeadsto the FirstDifferentiationEvents The Genomesof Most SomaticCellsAre Complete

Cussrc ExprRlueruT 22.1 UsingLethal

961 961

SignalGradientsMay InduceDifferent Cell Fates

963

SignalAntagonistsInfluenceCell Fatesand Tissue Induction

965

A Cascadeof SignalsDistinguishes Left from Right

956

.

coNTENTs

999

960

GastrulationCreatesMultiple TissueLayers,Which BecomePolarized

xxxiv

Mutationsto Study Development

959

23 NERVE CELLS !fl

Neurons andGlia:Building Blocksof the NervousSystem

1001

lOOz

Information FlowsThrough Neuronsfrom Dendrites to Axons 1003

NXXX

.

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u o ll) u n l p u e a r n lr n r ls :su lln q o l6 0 u nuul

SpecificMutations TransformCulturedCells into Tumor Cells

1113

A Multi-hit Model of CancerInduction ls Supported by SeveralLinesof Evidence

1114

Successive OncogenicMutations Can Be Traced in Colon Cancers

1116

PatternsCan DNA MicroarrayAnalysisof Expression RevealSubtle DifferencesBetweenTumor Cells

1116

@ Th e

G e ne ti cB a si so f ca n ce r

1119

Gai n-of-FunctionM utationsConvertProto-oncogenes 1119 into Oncogenes VirusesContainOncogenesor Cancer-Causing Activate CellularProto-oncogenes

1121

Mutations in Tumor-Suppressor Loss-of-Function GenesAre Oncogenic

1123

Genes Inherited Mutations in Tumor-Suppressor lncreaseCancerRisk

1123

Aberrationsin SignalingPathwaysThat Control DevelopmentAre Associatedwith Many Cancers 1124

!fl

oncogenicMutationsin GrowthPromoting Proteins

1127

OncogenicReceptorsCan Promote Proliferationin the Absenceof External Growth Factors

1127

Viral Activators of Growth-Factor ReceptorsAct as Oncoproteins

1128

Many OncogenesEncodeConstitutivelyActive Proteins Sig nal-Transduction

1129

InappropriateProductionof NuclearTranscription FactorsCan InduceTransformation

11 3 0

M o l e c u l a rC e l lB i o l o g yl s C h a n g i n gH o w C a n c e r ls Treated

1132

MutationsCausingLossof and Gr owth- lnhibiting Controls Cell-Cycle

1134

from MutationsThat Promote UnregulatedPassage Gr to 5 PhaseAre Oncogenic

1134

MutationsAffecting ChromatinLoss-of-Function RemodelingProteinsContributeto Tumors

1135

Lossof p53 Abolishesthe DNA-DamageCheckpoint 1136 Apoptotic GenesCan Functionas Proto-oncogenes 1137 Genes or Tumor-Suppressor CheckpointsOften Leadsto Failureof Cell-Cycle T u m o rC e l l s i n Aneuploidy

1138

and CaretakerGenes Carcinogens 11 3 9 in Cancer CarcinogensInduceCancerby DamagingDNA

11 3 9

SomeCarcinogensHave Been Linkedto Specific Cancers

1139

Lossof DNA-RepairSystemsCan Leadto Cancer

1141

Contributesto TelomeraseExpression lmmortalizationof CancerCells

1143

GLOSSARY

G-1 l-1

INDEX

CONTENTS

.

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'sJaJrloJpue seqeue 'so;n1lnl1s to sarloru ur eesuef a.rl sE Sur8ueqc-lseJe^eq pue dlprder e^ou aruos '(1-1 arn8r4) sadeqs pup sezrs lo f,lBrrcr, Surzeute ue ur otuoJ sllaJ

slla) Io f1r;euourruo)pue Alrsla^lqaql lU 's11arSurdpnts sdp.^a snorrel ar{l ruo{ urpel uef, to aA\ let{,ryrpup (suorlf,unJIeJrtrrr pup sluenlrlsuoJ Jrsgq Jroql 's11ec dlrs.rallp aqt Surcnporrurdq rerdeqr anSolord srql ;o ur truls elrN'slleJ rltpJp pue 'ayr1'qrrrg aqr 1o ,{lors petJceJ Jo -ulnru oqt 3ur,teo,ndgenper8 'splaryasegt to IIE r.uoryu,ry\pJp saqreo.rddelpluorurrodxapue slq8rsureqrrJsep11r,ue,tt 'sral -deqc SurrnolloJer{r uI 'uorletuour.radxa;o a1d1spue srseqd ',(8o1orq -rua u^\o slr spq splort eseql Jo qre1 leluarudolo,r -ep pue'eruerf,s relndruor tSolorsdqd'slrlaue8 1(docsonrur iiSoyorqrplnf,oloru'sorsdqdorq'dtlsruaqcorqreqtoSol sBur.rq (qcr.r sr lSolorq r lerll erualrs a,rrlBr8elut [e] JEInJOI6W 'puodsa; o1 sdem eleudordde tsotu oqt uo aprrep daqr moq pue sllor q8no.rqrs^{olJuorler.uro;ur^/rl,oq lnoqe dlrelnrrued 'peureal eg ol sureruoJlunotup asuerururue '11115 'Joqlo qJEe aJuenlturpu€ qrnot dagr ,vroq'urEluoJ daqt sernlcnrls tEr{na 's11ac sluouodruor ar{t tnoqp Etep.ry\au;ouorsoldxaue aJe! 1o o.tr 'suado d.rntuec tsrrJ-.{tue,v\teqr sV 'aJII Jo lrun Ietueruep 'aJIIyo sartradord>lreurller{ -unt oql sr IIar eqr tpr{t SuneJrpul aqt IIe trqrqxo susrueS.rorulnlloJrun aldurrsua,lg '1ac epurs e Jo lsrsuof, sulsrue8rodueru 1nq 'salnlf,n.rlsxalduror olur pezrve8rc sllel Jo suor1lrrtro suorllq ureluor srusrueSrore1 -NIIE]qFI.U

JOTIIO PUP EII

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(b) Eukaryoticcell

( a ) P r o k a r y o t icce l l P e r i p l a s m iscp a c e a n d c e l lw a l l

Golgi vesicles

Lysosome

O u t e rm e m b r a n e

I n n e r( p l a s m a ) membrane

Nucleoid lu'btrm I

Nucleoid

N u c l e a rm e m b r a n e P l a s m am e m b r a n e Golgi vesicles Mitochondrion Peroxisome Lysosome

I n n e r( p l a s m a )m e m b r a n e

Periplasmicspace Outer membrane R o u g he n d o p l a s m i c reticulum

1-2 Prokaryotic cellshavea simplerinternal A FIGURE micrograph of a organizationthan eukaryoticcells.(a)Electron coli a commonintestinal bacteriumThe thin sectionof Escherichia withina of the bacterial DNA,is notenclosed nucleoid, consisting by two membraneE coli andsomeotherbacterra aresurrounded spaceThethincellwallis membranes separated bythe periplasmic (b)Electron mrcrograph of a plasma adjacent to the innermembrane antibodies Onlya single cell,a typeof whitebloodcellthatsecretes (theplasma membrane membrane) surrounds thecell,butthe manymembrane-limited compartments, or interior contains Thedefining of eukaryotic cellsis organelles. characteristic

whichis DNAwithina definednucleus, of thecellular segregation is membrane Theouternuclear by a doublemembrane bounded for factory a reticulum, withthe roughendoplasmic continuous process andmodifyproteins, proteinsGolgivesicles assembling to digestcellmaterials generate lysosomes energy, mitochondria and process usingoxygen, molecules them,peroxisomes recycle them to the surfaceto release carrycellmaterials vesicles secretory E M u r r a y . P a r t ( b ) fromPC G B u r d e t t a n d R D J lPart(a)courtesyofl lJltrastructure: A Functional 1993,CellandTissue Cross andK L Mercer, andCompanyl W H Freeman Perspective,

and 40 miles up in the atmosphere;they are quite adaptable! The carbon stored in bacteria is nearly as much as the carbon stored in plants. Eukaryotic cells, unlike prokaryotic cells, contain a defined membrane-bound nucleus and extensive internal membranes that enclose other compartments called organelles (Figure 1-2b). The region of the cell lying betweenthe plasma membrane and the nucleus is the cytoplasm, comprising the cytosol (aqueousphase)and the organelles.Eukaryotescompriseall membersof the plant and animal kingdoms,including

the fungi, which exist in both multicellular forms (molds) and unicellular forms (yeasts),and the protozoans (proto, primitive zoan, animal), which are exclusively unicellular. Eukaryotic cells are commonly about 10-100 pm across' generally much larger than bacteria. A typical human fibroblast, a connective tissuecell, might be about 15 p,m acrosswith a volume and dry weight some thousandsof times those of an E. coli bacterial cell. An ameba, a single-celledprotozoan, can be more than 0.5 mm long. An ostrich egg beginsas a singlecell that is evenlarger and is easilyvisible to the naked eye. THE DIVERSITA Y N D C O M M O N A L I T YO F C E L L S

All cells are thought to have evolved from a common progenitor becausethe structures and moleculesin all cells have so many similarities. In recent years, detailed analysis of the DNA sequencesfrom a variety of prokaryotic organisms has revealed two distinct types: the bacteria and the archaea. Working on rhe assumption that organisms with more similar genesevolvedfrom a common progenitor more recently than those with more dissimilar genes,researchers have developed the evolutionary lineage tree shown in Figure 1-3. According to this tree, the archaeaand the eukaryotes diverged from bacteria billions of years ago before they diverged from each other. In addition to DNA sequence distinctions that define the three groups of organisms,

Animals

Plants Fungi

Ciliates

Euglena

Microsporidia

EUKARYOTA S I i m em o l d s Diplomonads (Giardia lamblia)

EUBACTERIA E. coli

Sulfolobus ARCHAEA B. subtilus

Thermococcus

Thermotoga

Methanobacteriu m Halococcus

Flavobacteria G r e e ns u l f u r bacteria

Halobacterium Methanococcus jannaschii

Borrelia burgdorferi

P r e s u m e dc o m m o n p r o g e n i t o r of all extant organisms P r e s u m e dc o m m o n p r o g e n i t o r of archaebacteria and eukaryotes

A FIGURE 1-3 All organismsfrom simplebacteriato complex mammalsprobablyevolvedfrom a common,single-celled progenitor.Thisfamilytreedepicts theevolutionary relations among thethreemajorlineages of organisms. Thestructure of thetreewas initially ascertained frommorphological criteria: creatures that look alikewereput closetogetherMorerecently the sequences of DNA andproteins havebeenexamined asa moreinformation-rich criterion for assigning relationships Thegreater thesimilarities in thesemacromolecular sequences, the moreclosely related organisms arethoughtto be.Thetreesbasedon morphological comparisons andthefossilrecordgenerally agreewellwith thosebasedon molecular data.Althoughallorganisms in the eubacterial and archaean lineages areprokaryotes, archaea aremoresimilar to eukaryotes ("true"bacteria) thanto eubacteria in somerespects For instance, archaean andeukaryotic genomes encodehomologous histone proteins, whichassociate with DNA;in contrast, bacteria lack histonesLikewise, the RNAandproteincomponents of archaean ribosomes aremorelikethosein eukaryotes thanthosein bacteria. C H A P T E RI

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archaeacell membraneshave chemical properties that differ dramatically from those of bacteria and eukaryotes. Many archaeansgrow in unusual,often extreme,environments that may resemblethe ancient conditions that existed when life first appeared on earth. For instance, halophiles ("salt loving") require high concentrationsof salt to survive, and thermoacidophiles("heat and acid loving") grow in hot (80' C) sulfur springs,where a pH of lessthan 2 is common. Still other archaeanslive in oxygen-free milieus and generate methane(CH+) by combining water with carbon dioxide.

U n i c e l l u l aO r r g a n i s m sH e l p a n d H u r t U s Bacteria and archaea, the most abundant single-celledorganisms,are commonly 1-2 pm in size. Despite their small size and simple architecture,they are remarkable biochemical factories,converting simple chemicalsinto complex biological molecules.Bacteria are critical to the earth's ecology, but some cause major diseases:bubonic plague (Black Death) from Yersiniapestis,strep throat from Streptomyces, tuberculosis from Mycobacterium tubercwlosis, anthrax from Bacillus anthracis, cholera from Vibrio cholerae, and food poisoning from certain types of E. coli and Salmonella. Humans are walking repositories of bacteria, as are all plants and animals.'We provide food and shelter for a staggering number of "bugs," with the greatestconcentration in our intestines.In return for the food and shelter that allow them to reproduce, bacteria help us digest our food. One common gut bacterium,E. coli, is also a favorite experimental organism. In responseto signalsfrom bacteria such as E. coli, the intestinal cells form appropriate shapesto provide a niche where bacteria can live, thus facilitating proper digestion by the combined efforts of the bacterial and the intestinal cells. Conversely,exposure to intestinal cells changes the properties of the bacteria so that they participare more effectivelyin human digestion. Such communication and responseis a common feature of cells. The normal, peacefulmutualism of humans and bacteria is sometimesviolated by one or both parties. When bacteria begin to grow where they are dangerousto us (e.g.,in the bloodstream or in a wound), the cells of our immune system fight back, neutralizingor devouring the intruders. Powerful antibiotic medicines,which selectivelypoison prokaryotic cells, provide rapid assistanceto our relatively slow-developingimmune response.Understanding the molecular biology of bacterial cells leads to an understanding of how bacteria are normally poisonedby antibiotics,how they becomeresistantto antibiotics, and what processesor structurespresent in bacterial but not human cells might be usefully targeted by new drugs. Like bacteria) protozoa are usually beneficial members of the food chain. They play key roles in the fertility of soil, controlling bacterial populations and excreting nitrogenous and phosphatecompounds,and are key playersin wastetreatment systems-both natural and man-made.These unicellular eukaryotesare also critical parts of marine ecosystems, consuming large quantities of phytoplankton and harboring photosynthetic algae, which use sunlight to produce biologically usefulenergyforms and small fuel molecules.

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FfGURE 1-4 Plasmodiumorganisms,the parasitesthat cause malaria,are single-celledprotozoanswith a remarkablelife cycle.ManyPlasmodium species areknown,andtheycaninfecta The hosts. varrety of animals, cycling betweeninsect andvertebrate fourspecies thatcausemalaria in humansundergo several dramatic (a)Diagram withintheirhumanandmosquito hosts. transformations of the lifecycleSporozoites entera humanhostwhenan infected Anopheles mosquitobitesa personfl Theymigrateto the liver, intothe wheretheydevelop intomerozoites, whicharereleased fromsporozoites, sothis bloodE. Merozortes differsubstantially "to transform" (Greek, isa metamorphosis or "many transformation merozoites invaderedbloodcells(RBCs) and shapes")Circulating producedby somePlasmodium reproduce withinthem B Proteins RBCs, causing the cellsto species moveto the surface of infected infected RBCs adhere to thewallsof bloodvessels. Thrsprevents fromcirculating to thespleen, wherecellsof the immunesystem theyharbor woulddestroy the RBCs andthe Plasmodium organisms in RBCs for a periodof time Aftergrowingandreproducing suddenly characteristic of eachPlasmodium species, the merozoites

from largenumbersof infectedcells4. lt burstforth in synchrony chillsthatare isthiseventthatbringson thefeversandshaking of malariaSomeof the released symptoms thewell-known a cycleof production creating RBCs, infectadditional merozoites intomaleand develop somemerozoites Eventually, andinfection. cells, These 5, anothermetamorphosis. femalegametocytes cannot whichcontainhalftheusualnumberof chromosomes, in bloodIo an Anopheles theyaretransferred for longunless survive gametocytes are the stomach, mosquito s In the mosquito. yetanother intospermor eggs(gametes), transformed flagellaon of longhairlike markedby development metamorphosis zygotesZ, of spermandeggsgenerates the sperm6 Fusion wallandgrowinto intothecellsof thestomach whichimplant of Rupture sporozoites for producing factories essentially oocysts, of sporozoites E; thesemigrateto the thousands an oocystreleases glands, settingthe stagefor infectionof anotherhuman salivary and of matureoocysts micrograph electron host.(b)Scanning stomach of sudace the external abut Oocysts sporozoites. emerging them that protects withina membrane wallcellsandareencased (b)courtesy of R E Sinden ] fromthe hostimmunesystem. lPart

However, some protozoa do give us grief: Entamoeba histolytica causesdysentery;Trichomonas uaginalis,vaginitis; and Trypanosoma brucei, sleeping sickness.Each year the deadliest of the protozoa, Plasmodium falciparum and related species,is the cause of more than 300 million new casesof malaria, a diseasethat kills 1.5 to 3 million people annually. These protozoans inhabit mammals and mosquitoes alternately,changing their morphology and behavior in responseto signalsin each of theseenvironments.They also recognize receptors on the surfacesof the cells they infect. The complex life cycle of Plasmodium dramatically illustrates how a singlecell can adapt to each new challenge it encounters(Figure 1-4). All of the transformations in cell type that occur during the Plasmodium Iife cycle are gov-

erned by instructions encodedin the geneticmaterial of this parasite and triggered by environmental inputs. The other group of single-celledeukaryotes, the yeasts, also have their good and bad points with respect to humans. as do their multicellular cousins, the molds. Yeasts and molds, which collectively constitute the fungi, have an important ecological role in breaking down plant and animal remains for reuse.They also make numerous antibiotics and are used in the manufacture of bread, beer,wine, and cheese.Not so pleasant are fungal diseases'which range from relatively innocuous skin infections such as jock itch and athlete's foot to life-threatening Pneumocystis carinii pneumonia, a common cause of death among AIDS patients. A N D C O M M O N A L I T YO F C E L L S THE DIVERSITY

VirusesAre the Ultimate Parasites

opportunity. Viral infections can be devastatingly destructive, causing cells to break open and tissuesto fall apart. However, many methods for manipulating cells depend on using virusesto convey geneticmaterial into cells.To do this, the portion of the viral genetic material that is potentially harmful is replaced with other genetic material, including human genes.The altered viruses, or vectors, still can enter cellstoting the introduced geneswith them (Chapter 9). One day, diseasescausedby defectivegenesmay be treated by using viral vectors to introduce a normal copy of a defective gene into patients. Current research is dedicated to overcoming the considerableobstaclesto this approach, such as getting the introduced genesto work at the right placesand trmes.

Not all microscopic pathogensare cells. The other most familiar disease-causingorganisms are the viruses, which make use of the machinery inside the cellsthey infect to copy themselves.Virus-causeddiseasesare numerous and all too familiar: chickenpox, influenza, some types of pneumonia, polio, measles,rabies,hepatitis,the common cold, and many others. Smallpox, once a worldwide scourge,was eradicated by a decade-longglobal immunization effort beginning in the mid-1960s. Mral infections in plants (e.g.,dwarf mosaic virus in corn) have a major economic impact on crop production. Planting of virus-resistantvarieties, developed by traditional breeding methods and more recently by geneticengineeringtechniques,can reduce crop lossessignificantly. Most viruses have a rather limited host range, infecting certain bacteria,plants, or animals(Figure1-5). Becausevirusescannot grow or reproduce on their own, they are in this sensenot consideredto be alive. To survive. a virus must infect a host cell and take over its internal machinery to synthesizeviral proteins and in some casesreplicate the viral geneticmaterial. When newly made virusesare releasedby budding from the cell membrane or when the infected cell bursts, the cycle starts anew. Viruses are much smaller than cells,on the order of 100 nanometer (nm) in diameter; in comparison, bacterial cells are usually >1000 nm (1 nm : 10-e meters).A virus is typically composedof a protein coat that enclosesa core containing the genetic material, which carries the information for producing more viruses (Chapter 4). The coat protects a virus from the environment and allows it to stick to, or enter,specifichost cells. In some viruses, the protein coat is surrounded by an outer membrane-likeenvelope. The ability of viruses to rransport genetic material into cells and tissuesrepresentsa medical menaceand a medical

The most remarkable feature of organismsis their ability to reproduce.Biological reproduction, combined with continuing evolutionary selectionfor a highly functional body plan, is why today's horseshoecrabs look much as they did 300 million years ago, a time span during which entire mountain ranges have risen or fallen. The Teton Mountains in S7yoming,now about 14,000 feet high and still growing, did not exist a mere 10 million years ago. Yet horseshoecrabs, with a life span of about 19 years,have faithfully reproduced their ancient selvesmore than half a million times during that period. The common impression that biological structure is transient and geologicalstructure is stableis the exact opposite of the truth. Despite the limited duration of our individual lives, reproduction gives us a potential for immortality that a mountain or a rock does not have. Whereas some species have changed little over great periods of time, other organismshave changeddramatically

(a)T4 bacteriophage

(b)Tobaccomosaicvirus

C h a n g e si n C e l l sU n d e r l i eE v o l u t i o n

,

50nm

,

(c) Adenovirus

A FIGURE 1-5 Virusesmust infect a host cell to grow and reproduce.Theseelectronmicrographs illustrate someof the structural variety (a)T4 bacteriophage exhibited byviruses. (bracket) attaches to a bacterial cellviaa tailstructureViruses that infect bacteria arecalledbacteriophages, (b)Tobacco or simplyphages. mosarc viruscauses a mottlingof the leaves of infected tobacco C H A P T E R1

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plantsandstuntstherrgrowth.(c)Adenovirus causes eyeand respiratory tractrnfections in humans. Thisvirushasan outer membranous envelope fromwhichlongglycoprotein spikesprotrude [Part(a) from A Levine,1991, Viruses,ScientificAmericanLibrary,p 20 Part(b) courtesyof R C Valentine Part(c) courtesyof RobleyC Williams, Universityof Californial

during the same period. The changescame in responseto pressuresfrom the environment that causedincreasedsurvival of variant individuals. Both stasis and change are possible becausethe machinery of cells does an amazingly precisejob of copying geneticmaterial, yet its rare errors introduce some variation. If environmental conditions continue to select more or less the existing form, as in the caseof horseshoecrabs, the specieswill changelittle. If a new variant has a survival advantage, perhaps because conditions have changed, it may persist and replace the old form. Populations of bacteria exposedto antibiotics, for example, changetheir properties dramatically to escapeand live. They do this becauserare mutations, changesin the genetic material that allow antibiotic resistance,keep some cells alive while the cells without those mutations die. Most populations of any single specieshave a large repertoire of genetic alterations becausethere is a low but significant error rate in copying the genes.That error rate increasesin the presenceof radiation such as sunlight or certain chemical poisons. Current genomeproiects are exploring geneticvariation among humans. "The" human genome sequencethat has been determined already is just one version among billions. Understanding variation is essentialto learn how we respond differently to certain infections or drugs and to exploring how our geneticheritage combines with our experienceand learning to make each of us unique. Underlying the reproduction of organismsis the copying of cells, something that must be precise in order to control the size, shape, and organization of animals and to prevent unwanted growth, such as cancer.The cell is a machine that can copy itself, unlike viruses,which cannot do so on their own. As we will seein Chapters 20 and 21',the cell cyclefrom a single cell copying its own contents through division into two cells-is controlled by a seriesof elegant switches and cross-checking mechanisms. Reproducing cells accurately is a matter of life and death.

EvenSingleCellsCan Have Sex If genetic material was never shared or exchanged,each individual would be the beginning of a new clone of individuals, and the members of a clone would share most of the same genetic strengths and weaknesses.Sex is a processof mingling genetic variation from two individuals, creating new individuals with a combination of properties unlike either parent and that may be beneficial for survival and reproduction. Each chromosome except the sex chromosomes is representedtwice, one copy from the father and one from the mother. Since each pair of chromosomes trades piecesduring the formation of eggs and sperm, new combinations of genesare created and inherited together in the next generation-variation is accelerated. The other benefit of having two copiesof each chromosome is that a poorly functioning gene is backed up by the other copy. The common yeast used to make bread and beer, Saccharomycescereuisiae,appearsfairly frequently in this

Budding lS. cerevisi ael

1-6 The yeast Saccharomycescerevisraereproduces A FfGURE sexuallyand asexually'(a)Twocellsthat differin matingtype' calleda andcr,canmateto forman a/a cellIl. Thea anda cellsare theycontaina singlecopyof eachyeast meaning haploid, halfthe usualnumber'Matingyieldsa diploida/ctcell chromosome, Duringvegetative two containing copiesof eachchromosome. process an asexual by mitoticbudding, growth,diploidcellsmultiply a diploidcellsundergomeiosis, conditions, E Understarvation B. Rupture to formhaploidascospores typeof celldivision, special into whichcangerminate fourhaploidspores, releases of an ascus asexually E. (b)Scanning alsocanmultiply cells4. These haploid Aftereachbudbreaks of buddingyeastcells. micrograph electron buds free,a scaris left at the buddingsite,so the numberof previous (b) AbbeyA/isuals M bacteria are cells [Part canbe counted.Theorange lncl Unlimited, book becauseit has proven to be a great experimental organism. Like many other unicellular organisms' yeasts h"u. t*o mating types that are conceptually like the male and female gametes(eggsand sperm) of higher organisms'

ubiquitous. A N D C O M M O N A L I T YO F C E L L S THE DIVERSITY

.

7

We Developfrom a SingleCell In 1827, German physician Karl von Baer discovered that mammals grow from eggs that come from the mother's ovary. Fertilization of an egg by a sperm cell yields a zygote, a visually unimpressive cell 200 pm in diameter. Every human being begins as a zygote,which housesall the necessaryinstructions for building a human body containing about 100 trillion (1014)cells, an amazing feat. Development begins with the fertrlizedegg cell dividing into two, four, then eight cells, forming the very early embryo (Figure 1-7). Continued cell proliferation and then differentiation

Chapters 16 and 22. Making different kinds of cells-muscle, skin, bone, neuron, blood cells-is not enough to produce the human body. The cells must be properly arranged and organized into tissues,organs, and appendages.Our two hands have the same kinds of cells, yet their different arrangements-in a mirror image-are critical for function. In addition, many cells exhibit distinct functional and/or structural asymmetries, a property often called polarity. From such polarized cells arise asymmetric, polarized tissuessuch as the lining of the intestinesand structures such as hands and hearts. The features that make some cellspolarized and how they arise also are coveredin later chapters,including Chapter 21.

Video:EarlyEmbryonicDevelopment < FIGURE 1-7 The first few cell divisionsof a fertilized egg set the stagefor all subsequentdevelopment.A developing mouseembryois shownat (a)the two-cell,(b) four-cell, and(c)eight-cell stages. Theembryoissurrounded by supporting membranes. The corresponding stepsin human development occurduringthe firstfew daysafterfertilization. Researchers. IClaudeEdelmann/Photo I n cl

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Stem Cells,Fundamentalto FormingTissues and Organs,Offer MedicalOpportunities The biology of stem cells, cells that can give rise to specific cell types and tissues,has generated, great interest. 'We can 'Sfhen contrast stem cells to the simpler types of bacteria. a bacterial E. coli cell divides, both daughter cells are pretty much equivalent in content, size, and shape. In some other bacteria and in many casesof eukaryotic cell division, the two daughters differ in important ways. Although both will have the same genetic material, the cells may differ in size, shape, and contents. The cells may have different fates, that is, they may become different types of differentiated cell. A division that produces two different daughter cells is sometimes described as an asymmetric cell division. Stem-celldivisions are a specialcaseof asymmetric division. One of the two daughter cells is identical to the parent cell; the other follows a path of differentiation, such as becoming a blood cell. The parent cell, called a stem cell, can go on reproducing itself at every division, at each division also producing anorher blood cell. Mosr tissuesin our bodies form from stem cells. Blood, for example, is produced from stem cells that residein the bone marrow and continue to produce new blood cellsfor our entire lives. This is the basis of the often successfulbone marrow transplants that are used to treat cancer patients who have had their blood stem cells damaged by cancer treatments: what is being transplanted is stem cells.However, blood stem cells produce onlv more of themselvesand blood cells, not othir cell types. Thus eachtissuemust have its own stem cells,at leastduring the period of development when the tissue is formed. Stem cells for each tissue arise from even more capable stem cells that have the ability to form multiple stem cell types. The first stem cells are found in early embryos,where all the cells are capable of producing all cell types. In mammals the ultimate stem cell is the fertilized egg, which produces early embryo cells capable of forming all the tissuesof the body. This capability is illustrated by the formation of identical twins, which occur naturally when the mass of cells composing an early embryo divides into two parts, each of which develops and grows into an individual animal. This means that the cells cannor have divided up their embryo-forming duties prior to the time of embryo division. Each cell in an eight-cell-stagemouse embryo has the potential to give rise to any part of the entire animal. Cells with this capability are referred to as embryonic stem (ES)cells. As we will learn in Chapter 22, ES cells can be grown in the laboratory (cultured) and will develop into various types of differentiated cells under appropriate conditions. The ability to make and manipulate mammalian embryos in the laboratory has led to new medical opportunities as well as various social and ethical concerns.In vitro fertilization, for instance, has allowed many otherwise infertile couples to have children. One technique involves extractron of nuclei from defective sperm incapable of normally fertilizing an egg,injection of the nuclei into eggs,and implantation of the resulting fertilized eggsinto the mother.

In recent years, nuclei taken from cells of adult animals have been used to produce new animals. In this procedure' the nucleus is removed from a body cell (e.g., skin or blood cell) of a donor animal and introduced into an unfertilized mammalian egg that has beendeprived of its own nucleus.In a step that has now been done with mice, cows, sheep, mules, and some other animals, the egg with its donor nucleus is implanted into a foster mother. The ability of such a donor nucleusto direct the developmentof an entire animal shows that all the information required for life is retained in the nuclei of some adult cells. Sinceall the cells in an animal produced in this way have the genesof the single original donor cell, the new animal is a genetic clone of the donor (Figure 1-8), though the animals may differ anyway due to their distinct environments and experiences.Repeating the processcan give rise to many clones. Nuclei taken from ES cellswork especiallywell, whereasnuclei from other parts of the body at later times in life work far lesswell. The majority of embryos produced by this technique do not survive due to birth defects,so the donor nuclei may not have all the needed information or the nuclei may be damaged by the cloning process.Even those animals that are born alive have abnormalities,including acceleratedaging. The "rooting" of plants, in contrast, is a type of cloning that is readily accomplished by gardeners,farmers, and laboratory technicians. Scientific interest in the cloning of humans is very limited. Virtually all scientistsoppose it becauseof its high risk to the embryo (also, most people don't believe there is a

critical shortage of twins and triplets). Of much gteater scientific and medical interest is the ability to generatespecific cell types starting from embryonic or adult stem cells' This procedure, somatic cell nuclear transfer (SCNT)' produces cells that are grown in culture and never turned into

If the cells are produced using a donor nucleus from a patient, the properties of the cells may allow them to escaperejection by the patient's immune system,opening new possibilities for cell-transplanttherapies.

E

The Moleculesof a Cell

Molecular cell biologists explore how all the remarkable

ture and function.

Small MoleculesCarryEnergy,TransmitSignals, and Are Linkedinto Macromolecules Much of the cell's content is a watery soup flavored with small molecules (e.g., simple sugars' amino acids, vitamins) and ions (e.g.,sodium, chloride, calcium ions)' The locations conce.tirations of small molecules and ions within the "nd controlled by numerous proteins inserted in cellular are cell membranes. These pumps' transporters, and ion channels move nearly all small moleculesand ions into or out of the cell and its organelles(Chapter 11). One of the best-known small molecules is adenosine triphosphate (ATP), which storesreadily available chemical 'When ..r"rgy itt two of its chemical bonds (seeFigure 2-31)' cells"splitapart theseenergy-richbonds in AIP' the released ..r.rgy ."n b. harnessed to power an energy-requiring pro..tt such as muscle contraction or protein biosynthesis' 1-8 Fivegeneticallyidenticalclonedsheep.An early To obtain energy for making ATR cells break down food a FIGURE intofivegroupsof cellsandeachwas sheepembryowasdivided molecules.For instance, when sugar is degraded to carbon natural like the much mother, intoa surrogate implanted separately dioxide and water, the energy stored in the original chemical process of twinningAt an earlystagethecellsareableto adjustand bonds is releasedand much of it can be "captured" in ATP the cellsbecome laterin development forman entireanimal; (Chapter 72).Bacterial,plant, and animal cells can all make way andcanno longerdo so.An alternative progressively restricted ATP ty this process.In addition, plants and a few other orsingle-celled the nucleiof multiple isto replace to cloneanimals ganisms can harvest energy from sunlight to form ATP in with donornucleifromcellsof an adultsheep'Eachembryo embryos photosynthesis. ' was to the adultfromwhichthe nucleus identical will be genetically Other small moleculesact as signalsboth within and beto theseprocedures survive of embryos obtained.Lowpercentages tween cells; such signals direct numerous cellular activities on the givehealthy andthefull impactof the techniques animals, effect on our bodies of Library/Photo (Chapters iS uttd f e ;. fne powerful Photo Tompkinson/Science animalsis not yet known.lGeoff flooding of instantaneous the from comes a frightening event Incl Researchers, S F A CELL T H E M O L E C U L EO

O

9

the body with epinephrine, a small-moleculehormone that mobilizes the "fight-or-flight" response.The movements neededto fight or flee are triggered by nerve impulses that flow from the brain to our muscleswith the aid of neurotransmitters, another type of small-moleculesignal that we discussin Chapter23.

acids. Sugars,for example, are the monomers used to form polysaccharides. These macromolecules are critical structural components of plant cell walls and insect skeletons.A typical polysaccharideis a linear or branched chain of repeating identical sugar units. Such a chain carries information: the number of units. However, if the units arenot identical, then the order and type of units carry additional information. As we will seein Chapter 6, some polysaccharides exhibit the greaterinformational complexity associated with a linear code made up of different units assembledin a particular order. But this property is most typical of the two other types of biological macromolecules-proteins and nucleic acids.

ProteinsGive CellsStructureand perform Most CellularTasks The varied, intricate structures of proteins enable them to carry out numerous functions. Cells string together Z0 dif_ ferent amino acids in a linear chain to foim a protein (see Figure 2-14).Proteins commonly range in length from 100

to 1000 amino acids, but some are much shorter and others longer. We obtain amino acids either by synthesizingthem from other moleculesor by breaking down proteins that we eat. The "essential" amino acids, from a dietary standpoint, are the eight that we cannot synthesizeand must obtain from food. Beans and corn together have all eight, making their combination particularly nutritious. Once a chain of amino acids is formed, it folds into a complex shape,conferring a distinctive three-dimensionalstructure and function on each protein (Figure1-9). Some proteins are similar to one another and therefore can be consideredmembers of a protein family. A few hundred such families have been identified. Most proreins are designedto work in particular places within a cell or to be releasedinto the extracellular (extra, "outside") space.Elaborate cellular pathways ensurethat proteins are transported to their proper intracellular (intra, "within") locations or secreted (Chapters 13 and14). Proteins can serveas structural componentsof a cell. for example,by forming an internal skeleton(Chapters tO, tZ, and 18). They can be sensorsthat changeshapeas remperature, ion concentrations, or other properties of the cell change. They can import and export substancesacross the plasmamembrane(Chapter11). They can be enzymes,causing chemicalreactionsto occur much more rapidly than they would without the aid of theseprotein catalysts(Chapter 3). They can bind to a specificgene,turning it on or off (Chapter 7). They can be extracellular signals,releasedfrom one cell to communicatewith other cells, or intracellular signals, carrying information within the cell (Chapters 15 and 16). They can be motors that move other moleculesaround, burning chemicalenergy (ATP) to do so (Chapters17 and 1g).

Insulin

Glutaminesynthetase Lrturamtne synthetase

Hemoglobin

FIGURE 1-9 Proteinsvary greatly in size,shape,and function.Thesemodels of thewater-accessible surface of some representative proteins aredrawnto a commonscaleandreveal the numerous projections andcrevices on thesurface. Eachprotern hasa definedthree-dimensional shape(conformation) thatisstabilized by numerous chemrcal interactions discussed in Chapters 2 and3. The illustrated proteins include (glutamine enzymes synthetase and

10

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L | F EB E G T N W S |TH CELLS

DNA molecule

lmmunoglobulin

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adenylatekinase),an antibody(immunoglobuiln), a hormone (insulin), and the bloods oxygencarrier(hemoglobin)Modelsof a segmentof the nucleicacid DNA and a smallregionof the lipid bilayerthat formscellularmembranes(seeSection1.3)demonstrate the relativewidth of thesestructures comparedwith that of tvpical proteins.[Courtesy of GarethWhite]

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'g rardeq3 ur lretepur uortElsuertpuB uorldrrrsuPrl lno d.rrel leqt stuauodruor IIer eql euruexe al1 'opof, f,rteu -aE les.ra.trundlreeu aql ot Surprorre aruanbasVNUIU oqr dq parelrrp rap.ro asne;d eqt ur sprre ourrue raglaSol s>1ur1 (uorlelsuB-rl SurrnC 'uorlelsueJl pue selqtuesseatuosoqrrar{l pell€r <sserord puocaseqt lno srrJrer'uralord pue VNU qtoq yo pasodruor eurqJprurelnJelou xalduoc ,{lsnourrouaue 'aruosogrraLIteJeH 'ruseldoldceql ol se,l.oruqcrq.u 'a1nca1ou (VNUru) y51A ra8uasserurelleruse otur possarordsr rrnpord 'sl1ar 'arelduer E se ruodre>1na u1 vNd IBrlrur aqr vNC Sursnureqr VNU p otur saprloe]f,nuJo a8e>1ur1 aql sez.{1etec 'au.,(zua 'VNC 'aserorudlod y51g e8rel pepuens-alqnop V eql Jo uorsre^ (VNU) prJp Jrelf,nuoqrrpepuerts-a13urs e otur pardor sr aua8 e yo uor8er Surpor eqt 'uorldrrcsuprl pellel 'rs-rr; '(II-I aqt uI arn8lg) suretordorur VNC ur uorlerurot -ur pepol aqt ol sorrasur sessecordo.la.lesn slla] lJeluor 'opuru sr uratord Jr{t sllal qlrr{.a uI puE uel{,{\ slorluof, teqt uo8al Ktolap8at E pue urelord e ;o acuanb -es prf,p ounue aqt sarpads rcq7 uot8at Surpoc e :strud orrrl ureluoJ dluorutuoc suralord 3ur1eu Jot suorlfnJlsur d.r.rer teqr saue8eql'000'SZ-000'02 lnoqe'suerunq lsaue8pues -noqt .,taale e^eq errolf,pq tso14 '3uo1 seprloallnu 000'00I or 000S a.re dlyecrddrqlrq^\ 'souo8 eql (strun leuorlrunt elerrslP olul poPI^lPsl VNq Jo uorlrod Surreaq-uotlEturolul oql 'puerts e 3uo1esoprloalJnuJo rapro reaurl Jrll 'aluenb -esslr ur seprser VNC dq perrrpr uorteruroturlrleueg or{J. 'gfleru lou oP ]Pql sPuprls ror{rodueu sureluotroslEuorl vNC -nlos oqt ue^a 'Surraddrzot peal 'r llr.{\ puerts dretuauralduroc rel{lo eqt Surureluocuortnlos e ur raded aql Suopos taded 'alduruxerod ;o aoarde o] Paqleue pue par;rrndsI puerls auo;t reqlo aql Sursnpue.rtseuo Surlcalaprot Intesndlauarlxa sruorl -enpuq;1.i:rl prf,e f,rolf,nur.lrns 'suorlrpuoo arnle.radtuetpue tles drz dlsnoauetuods11,udaqt 'pale.r rq8rr eqr ur raqtaSor 4ceq -edes a.le spuprts .,(reluerualduol teqt Suols os sr spuerls Jr o.,rueqr;o Surqrreru,{-ruluaualduol srr{J '(91-1 arn8r4)3 e qtr.&\peqlteru sr J qrea pue 1 e ser1puerls reqlo eqt 'y ue seq pupJls ouo Jeleror{,la.:uouJnrlsuof aydrursp spr{ x[eq elqnop 'puens rqr VNC r{leg to auoqlleq lerqeq eqr tuo4 lno Sunoal 'puerts -ord stred eseq eqt qtrlr E ur puo ol pua paurol VNCI are '9 pue 'J t'V peter^argqe'seprtoalcnuluora11rp rnoC

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a)eld pue ourrl lq6tu aql le surelord6ur4ey1ro; uolleturo+ulpapo) f.r.re1sp!)v )rel)nN '(solnralou 80I x S) utlle utotord IErnltnDS lu€punqE eqr or (salncalotu000'02) utalo.rdroldoc 'dleprm -a.r sutetord alrnb eqt uo.r; sarrB,r Surpurq-urlnsur erpr 'e8e.ra,reuo urolord tuere;trp Jo eruepunqe aql 'lce1 u1 1o addr qree Jo selnrelouruorlpu e ot osolf,suleluol IIOJE snqr isurelord ruere;Jrp000'0I rnoqe suleluor IIat re^II p leq] raprsuoJ taqrrny dels auo uollelnf,lef, srqt drrec oI '(Ez0I x ZO'9) punoduor lerlueqr due 1o ayourrad salnrelotu Jo requnu eql laqunu s,orpeSorrypue lq8ra,t uratord Ielol eqt trrorl sp IIer re^II .radsalncalou utarord 60I x 6'L lnoqe 'suratord f,rlofre>lna to reqrunu letor oqt elelnllel uer e,l.r '(1ou73) 00L'TS yo rq8rorr ;o lerrddr sr enlE^ srql Surunssy relnrelorrr p seq uratord lseef e8erarreeql '3 or_0I x L er{r 'tq8ta,trs(ller e to tuer sr ureto.rdrelnller rq8rarvr ;o I€tol -nd 97,,(laleturxo.rddpro; slunoJJe uralord erur5 '3 r_0I x g'g q8ra.uplno.vi aql'1uF E0'I Jo &lsuep 11ec e Sururns IIer -sy '(s;arqqlur ro) etunlol e seg 'aprse e*l o_0I x V't p srql '(1ac re,rr1) uo (tuc rurl eqnr E ,{1q3nor'11ac SI SI00'0) 'req ardcoledeqe se r1rns'11accrlodre>1ne lerrd& e a4et s.ta1 -unu srqt eterurtseoJ 'lasrr urelureru pue alerado ol spJJu IIar e selnrelotu urarord dueur .uoq >1serq8nu a^.\txaN 'dtlulJul 3uo1e pre,&ol 1a,'nsr sutetord algrssodIo reqrunu .ro'alqetsun'luele.trnba llleuorr eqt'elqElunorsrpasr,&\rar.{lo 'seruanb -lunJ dueru aq asaql Surunsse ua,tg p1no.l.r leqt Jo '3uo1 sprre -as uralord luereJJrp alqrssod oov17ere araqt ounue 00t rnoqe sr ureto.rd..1errddl,,E Jr lng 'ecue13lsrr; re alqrssodrursrueestI is>lserperre^ eseqlurotred or papaeu surolord ruereJtrpoqt IIE ruroJ splre ouIIuE 0Z uel ,{\oH

ur pegrrtsap are aruosrldarer{r Jo sluauodruol raqto dueru aql lpue;rs VNC € otur seprtoepnu Surlury ro; alqrsuodsa_r sr eserau,(1odVNq 'leur8r.roeql ot Iefrluepr ql€e ,serrlaq elqnop 1o ;red e sr eruortno eqI .(0I-I a.rn8rgaas)pue_us dreruetualduoJ 1y\eue olur saprloopnu elquesse ol aleldual e se qrea sasnpup seuosoruorql eqr ul vNC Ielrleq PuErls -elqnop spuertso.,vrtrqt seteredas,aruosrlde.r eqt (eurqrpru Jo uorlerrlda.ruralordrllnru a8rel e ,saprlrp 11ace arurl d.ra,rg 'pec ol 'JlpLlreqlo eq.l 'asaql !tuoy41o1 IlEq peJen og UEJ'seue8eqt;o JIeq snLlrpue '(71-1 ern8r4)sauosoruorqr seq uerunq Jo 'lpH 9t IIal lpur -rou dre,te 'rulads pue s33a uondarxe egr qtrl1 .VNC 1o to luerualduror eJrluastr sasr.rduorrusrue8roue ;o auroua8eq1 '(e7-1 a-rn8r1 aes) aql yo uor8arI€rtueJaqt ur ,saurt duetu 11ac 'sat1alncalorusrqr lq13ua1ur ralaurllru Jlesrluo >lrEqPePIoJ B tnoqE alnrelotu VNC relnrrrr a13ursB ur sJprseruorterurot 's11arrrtodrelord u1 'suretord -ut cttaue83qt to IIe ro tsoru urEtJaJqtr.4apelerJosseelnfelou VNC Jeeurl a13urse surel -uor aruosoruonll qleg '(9 ;erdeq3) saruosoruorrlf,se.{\ou{ J^\ soJnlJnrls JprlrrueJor.[l olur peplot dle,rrsuelxa ,snap -nu agl ur petef,ol sr sllJJ lrodrelna ul VNC er.[]Jo rsory

u o t s r ^ t ol l o ) 6 u u n c p o l e ) r l d o up u e saurosotuotq)olur pa6e{)ed sl oruouagoql 'paroldxa Surag ere uorteln8e.r 11rrs ,saue8 1o ed& srql Jo &rnbrqn pue sursrupq)au eql qSnoql II€ ro tsoru alelnSer deu sygg llerus seteturlsaaruos dg 'sldrrcsuerlauaS;o drrpqerspue uortrnpord ,uors Surtuln8er -sardxa aue8 uo tleJJef,rt€uerp p e^eq uel selnrelour VNU 'serurtrq8u aqr sllet le rq8rr aqr ur saua8tq8u or1tsale^ IIpruS -ItrE legt ualsds IortuoJ alrsrnbxaup ruroJ ol padoldruaa.re srolreJ uotldtnsue;l lueretlrp Jo sperpunq 'susruefiro xald -ruoJ ul 'seua8pereln8er aqr Suqcalasol dlpr;rceds Surpurq -VNC u,4o str Surtnquluoc uralord auo ueql JJou qlr.^A 'saxaldruor uleloJdrtlnu sE lJo,ln uouo sJolfet uortdr.rrsuerl ']as eqt ur aueSqrea rEau lsrxo rotret teqr roJ satrsSurpurq ;r seua8Jo las E eteln8er dlateurproor uer rotf,€J uortdr.rcs 'uorssarda;pue uortelrl -uert Jo addl auo 1o serdocaldrrlnyq '11ar -re eua8 e Jo drlclJlradseqt Suunsue ;o VNC aql ur rnl -lo e dlug .seprtoaltnu IIr.^aacuanbesrJf,nsqrpe 1o sardor.ra.a1 rnoJ Jo due aq uec uortrsodr1f,Ee erurs (9tS'gV1't) saruanb -as alqrssod o^eq uec s.rred espq 0I Sururetuor ylrlq 0rt '3uo1srred aseq y51q ;o ruaru8asy ZI-9 $oqe seouanbes t.roqs azruSoJerllr,l.r'uralord Surpurg-y51q e d1lecrdd1 'VNC s,ller e ut luaserdspuesnoqlJrll Jo rno seue8,ua1e tsnl 1o suor8a.rdroleln8ar aqr o1 dlyerluare;ardpurq ot elqp ere .{eql reql dlasnard os pedeqs are srotreJ uortdrrrsue{ '(1 retdeq3) saue8 relncrtred ;o uortdursuerl Surssard 'saqrlr,rs sE tJE pue -ar ;o SurtB,r.rlJe JerJlre VNC ot purq (srolreJuorldrrrsuprl qcrq,rrr pallEt suralordSurpurq-ypq uo spuadapdtr,rrlceaua8yo lortuoJ r{tns 'speeuluorrnr }eetu ol 'go surato.rd;o arrolrada.r rragl Surldepe ,(qareqr ro uo saue8 rrPeds Suturnt dq suortrpuof, ur se8ueqc.ro sleu8rs leuJetxe 'esrol aJrApue IeuJetxeot puodsa;ue) sllJJlueru taaoerory s11ecdaupr>1,{q peenpord tou er€ teqt suralord oruos ernp -ord s1larra,tq dq,rrr. s(teqJ 'sureto-rdaleu ot pesn pup (uo paurnt lo 'a,ttlf,eare saua8esoqtJo eruosdluo ad.{rIIac r{rea

s r r r ) H r M s N r g r sl j r ' r I

t u:ravul

zl

ur tnq 'seua3ueunq to tes IInJ aql uretuor serpogrno ur sllar eL[] IIE dl.reau'aruetsur roC 'paquf,suurl aq UEJ seua8rraql eJeq.4 pue uoq,lt IoJluoc ol sde,,vreleq s{usrup8ro 11y ' u r a t o r do t u l s J l n f , 'salncalotuy51a re8rel -elou VNUIU to uouplsuerr eqt pue (seLuosoluorqr Jo drlllqers eql Jo uollrunJ Pue erntf,nrts '3uo1 sepnoepnu 'sVNU aqr aleln8a.rd11err;roads 002-02 'dr{lqers pue Surssecord VNU pue ornl Ilprus sas€f,dueru u1 -fnrts eruosoruoJqlSurpnycurtlrrrrlre eua8 slf,odseduetu 1o Suneln8e.rur alor tuet.rodurr flqelreua; e Aeld ol puno' ueaq osle ser{VNU 'flruareg razrsaqludsuratord pue repear VNUIU luarrr]'a pue asrcard dlqe>1reua.rp a{pru ol surel -ord 69 ueqt eroril qlrrr,rdn rueat leqt sureqf,VNU JnoJ seq euosoqrr agt (oldurexa roC 'aurLlleru rplnlelou e Surplrnq ro; {ro^\euer; E se enres uer y51g 'useldoldc ol snolf,nu ruoJJ uorleruro;ur Surr.ra;supJlur aloJ slr ol uortrppe uI

ureqlrpourle olurspr)eouru.re 6ur1ur1 {1;erruaqr {q ureloLd e puearuenbes slrpear]eql sourosoqu {q punoqsr}r oloqm alqrlosse 'useldoyb y11y (VNUul) eq] o1 sa^oLr; rebuesseu ernleur oql 'llo) p ul :E de15saruanbas 6urporuou oAoLUar o1pessa:ord rrloi{re>1ne sr aq1: E dals eleldua]e sespuer]s ldursuerl VNCaq],o auo pepuerls-a16urs 6ursn]drnsuert e olurseprloapnu 6ur>1ur1 VNUU-ard esereuri;od aq_La]rsuelsoq] 'uorle)ol>rloeds VNc oq] 6uo;esenou.r 'VNC ue,Louorldursuerl sur6aq asereu{1od e ]e auebpale^r])e VNU aq]o] punoqxelduoruorlpr]rur ure1o.rdr1lnu e 1o{lquasse6urnnollo3 : g da15uJeq]ole^r]lepuelorluo),{eq}seue6:r;r:edsaq11o ,lo1eln6er aql o1purqsrolreluorldursue{:It da15'ssarord suor6el delsrllnu e {q surelold;o saruenbaspr)eourue oq} o}ul pouo^uo)s! vNc u! uollewrolu!papo)aqM-! lun9lJ v ureq3prce ouil..uv I 1 I I

v l g 1 o u o r o e ro u r p o c u o N V r u U ; ou o t 6 a l6 u t P o c - u t a 1 o l 3 vNc Jo uorOorpaqrrcsuerluoN -n2q2q2qa V N C + o u o r 0 o rp a q u 3 s u e {

uollolJcsue{

d uorleAllcv

can be "painted" for easy 1-12 Chromosomes FIGURE A normalhumanhas23 pairsof morphologically identification. fromthe onememberof eachpairis inherited chromosomes; distinct motherandthe othermemberfromthefather(Left)A chromosome whenthe froma humanbodycellmidwaythroughmitosis, spread wastreatedwith Thispreparation arefullycondensed chromosomes reaqents thatalloweachof the 22 pairs staininq fluorescent-labeled

Chapter 4. The molecular design of DNA and the remarkable properties of the replisome ensure rapid, highly accurate copying. Many DNA polymerasemoleculeswork in concert, each one copying part of a chromosome.The entire genome of fruit flies, about 1.2 x 10o nucleotideslong, can be copied in three minutes! Becauseof the accuracyof DNA replication, nearly all the cells in our bodies carry the same genetic instructions, and we can inherit Mom's brown hair and Dad's blue eyes. A rather dramatic example of genecontrol involves inac'Women tivation of an entire chromosomein human females. have two X chromosomes,whereas men have one X chromosome and one Y chromosome, which has different genes than the X chromosome. Yet the genes on the X chromosome must, for the most part, be equally active in female cells(XX) and male cells(XY). To achievethis balance'one of the X chromosomesin female cells is chemically modified and condensedinto a very small mass called a Barr body' which is inactive and never transcribed. Surprisingly,we inherit a small amount of geneticmaterial entirely and uniquely from our mothers. This is the circular DNA present in mitochondria' the organelles in eukaryotic cells that synthesizeAIP using the energy released by the breakdown of nutrients. Mitochondria contain multiple copies of their own DNA genomes,which code for some of the mitochondrialproteins(Chapter6). Becauseeachhuman inherits mitochondrial DNA only from his or her mother (it comes with the egg but not the sperm), the

colorwhen to appearin a different andtheX andY chromosomes of multiplex Thistechnique microscope viewedin a fluorescence (M-FISH) iscalled sometimes in situhybridization fluorescence (RiSht) fromthe (Chapter Chromosomes 6) painting chromosome orderof size, in pairsrndescending preparation on the leftarranged of X andY chromosomes Thepresence an arraycalleda karyotype. of M R Speicher] asmale [Courtesy the sexof the individual identifies

distinctive features of a particular mitochondrial DNA can be used to trace maternal history. Chloroplasts, the organelles that carry out photosynthesisin plants' also have Ih.i, o*tt circular genomes'Both mitochondria and chloroolasts are believedto be derived from endosymbionts,bacteii" th"t took up residenceinside eukaryotic cells in a mutually beneficialpartnership.The mitochondrial and chloroplast circular DNAs appeatto have originated as bacterial genomes' which also are usually circular,though the organellegenomes have lost most of the bacterialgenes.

Mutations May Be Good, Bad,or Indifferent Mistakes occasionallydo occur spontaneouslyduring DNA replication, causing changesin the sequenceof nucleotides' Soch ch"ng.s, or mutations, also can arise from radiation that causesdamageto the nucleotidechain or from chemical poisons, such as those in cigarette smoke, that lead to errors iuring the DNA-copying process (Chapter 25)' Mutations .o-.1., various forms: a simple swap of one nucleotide for another; the deletion, insertion, or inversion of one to millions of nucleotides in the DNA of one chromosome; and translocation of a stretch of DNA from one chromosome to another. In sexually reproducing animals such as ourselves,mutations can be inherited only if they are presentin cellsthat potentially contribute to the formation of offspring' Such germJine cells include eggs'sperm' and their precursor cells' T H E M O L E C U L EOSF A C E L L

O

13

Body cells that do not contribute to offspring are called somatic cells. Mutations that occur in these cells never are inherited, although they may contribute to the onset of cancer. Plants have a less distinct division between somatic and germ-line cells, since many plant cells can function in both capacltres. Mutated genesthat encode altered proteins or that cannot be controlled properly ."u.. .rurn..ous inherited diseases.For example, sickle-celldiseaseis atributable to a single nucleotide substitution in the hemoglobin gene, which encodesthe protein that carries oxygen in red blood cells. The single amino acid changecausedby the sickle cell muta-

Sequencingof the human genome has shown that a

transcription factors typically are only 70-12 nucleotides long, a few single-nucleotidemutations might convert a nonfunctional bit of DNA into a functional protein-binding regulatory site. Much of the nonessentialDNA in both eukaryotesand prokaryotes consistsof highly repeatedsequencesthat can move from one place in the genome to another. These mobile DNA elementscan jump (transpose)inro genes,most commonly damaging but sometimes activating them. Jumping generallyoccursrarely enoughto avoid endangering the host organism. Mobile elements,which *... di.coveredfirst in plants, are responsiblefor leaf color variegation and the diverse beautiful color patterns of Indian corn kernels.By jumping in and out of ge.resthat control prgmentationas plant developmentprogresses,the mobile elemenrsgive rise to elaboratecolorid putt...rr. Mobile elements were later found in bacteria, in which they often carry and, unfortunatelS disseminategenesfor antibiotic resrstance. Now we understand that mobile elements have multi_ plied and slowly accumulatedin genomesover evolutionary time, becoming a universal property of genomesin preseni_ day_organisms. They account for an astounding45 percent of the human genome. Some of our own mobile DNA ele_ ments are copies-often highly mutated and damaged_of genomesfrom viruses that spend part of their life iycle as DNA segmentsinserted into host-cell DNA. Thus we carry in our chromosomes the genetic residues of infections ac_ quired by our ancestors.Once viewedonly as molecularpar_ asites,mobile DNA elementsare now thoueht to have ion_ tnbuted significantly to the evolution of higher organisms (Chapter6). '14

.

cHAprE1 R | L | F EB E G t Nwsl r H c E L L s

IE

TheWorkof Cetts

In essence,any cell is simply a compartment with a watery interior that is separatedfrom the external environment by a surfacemembrane (the plasma membrane) that preventsthe free flow of moleculesin and out. In addition, as we've noted, eukaryotic cells have extensive internal membranes that further subdivide the cell into various comparrmenrs, the organelles.Each compartment has contents and properties, such as specializedproteins or a certain pH, suited to its job. The plasma membrane and other cellular membranes are composedprimarily of two layers of phospholipid molecules. These bipartite moleculeshave a "water-loving" (hydrophilic) end and a "water-hating" (hydrophobic) end. The two phospholipid layersof a membrane are orienred with all the hydrophilic ends directed toward the inner and outer surfacesand the hydrophobic ends buried within the interior (Figure1-13). Smalleramounts of other lipids, such as cholesterol, and many kinds of proteins are inserted into the phospholipid framework. The lipid moleculesand someproteins can float sidewisein the plane of the membrane,giving membranes a fluid character. This fluidity allows cells to change shape and even move. However, the attachment of some membrane proteins to other molecules inside or outside the cell restricts their lateral movement. We will learn more about membranes and how molecules cross them in Chapters10 and 11. The cytosol and the internal spacesof organellesdiffer from each other and from the cell exterior in terms of aciditg ionic composition, and protein contents. For example, the composition of saltsinside the cell is often drastically different from what is outside. Becauseof these different ,,microclimates," each cell compartment has its own assigned

Cholesterol

Water-seeking h e a dg r o u p

Water

FIGURE 1-13Thewatery interiorof cellsis surroundedby the plasmamembrane,a two-layeredshell of phospholipids. Thephospholipid molecules areoriented with theirfattyacylchains (blacksquiggly lines) facinginwardandtheirwater-seeking head groups(whitespheres) facingoutward.Thusbothsidesof the membrane arelinedby headgroups, mainlycharged phosphates, adjacent to thewateryspaces tnside andoutside thecell.All biological membranes havethesamebasicphospholipid bilayer structureCholesterol (red)andvarious (notshown)are proteins embedded in the bilayer. Theinteriorspaceisactually muchlarger relative to thevolumeof the plasma membrane depicted here.

tasks in the overall work of the cell (Chapters 10, L2, and 13). The unique functions and microclimates of the various cell compartm€nts are due largely to the proteins that reside in their membranesor interior. 'We can think of the entire cell compartment as a factory dedicatedto sustainingthe well-being of the cell. Much cellular work is performed by molecular machines, some housed in the cytosol, some attached to the cytoskeleton' and some in various organelles.Here we quickly review the major tasks that cells carry out in their pursuit of the good life.

Cells needto break down worn-out or obsoleteparts into small molecules that can be discarded or recycled' This housekeepingtask is assignedlargely to lysosomes,organelles crammedwith degradativeenzymes.The interior of a lysosome has a pH of about 5.0' roughly 100 times more acidic than that of the surrounding cytosol. This aids in the breakdown of materials by lysosomal enzymes' which are specially

CellsBuild and DegradeNumerous Moleculesand Structures As chemical factories,cells produce an enormous number of complex molecules from simple chemical building blocks. All of this syntheticwork is powered by chemical energy extracted primarily from sugarsand fats or sunlight, in the case of plant cells, and stored primarily in ATP, the universal "currency" of chemical energy (Figure 1-14). In animal and plant cells, most ATP is produced by large molecular machines located in two organelles,mitochondria and chloroplasts. Similar machines for generating ATP are located in the plasma membrane of bacterial cells. Both mitochondria and chloroplasts are thought to have originated as bacteria that took up residenceinside eukaryotic cells and then became welcome collaborators (Chapter 12). Directly or indirectlS all of our food is created by plant cells using sunlight to build complex macromolecules during photosynthesis. Even underground oil suppliesare derived from the decay of plant material.

llll+ overviewAnimation:BiologicalE

Most of the structural and functional properties of cells de-

cell and most membrane proteins, however' are made on ribosomes associatedwith the endoplasmic reticulum (ER)' This organelleproduces,processes'and ships out both proteins and lipids. Prolein chains produced on the ER move to the Golgi comple*, where they are further modified before being for*ari.d to their final destinations. Proteins that travel in this way contain short sequencesof amino acids or attached sugar ch"itts (oligosaccharides)that serve as addressesfor directing them to their correct destinations. These addresseswork becausethey arerecognizedand bound by other proteins that do the sorting and shipping in various cell compartments'

lnterconversions or Light (photosynthesis) compoundswith high potentialenergY(resPiration )

Synthesisof cellularmacromolecules(DNA, RNA, proteins, polysaccharides)

Synthesisof other cellularconstituents (suchas membrane phospholipidsand certainrequired metabolites)

Cellularmovements, includingmusclecontraction,crawling movements of entire cells, and movement of chromosomesduring mitosis

A FIGURE1-14 ATP is the most common molecule used by cells to capture and transfer energy. ATPis formed from ADP and in plantsand by the inorganicphosphate(Pi)by photosynthesis

Transportof moleculesagainst a concentration

gradient

Generationof an electricpotential acrossa memDrane

Heat

for nerve 1fi..:.,;t;,",

by andfatsin mostcells.Theenergyreleased of sugars breakdown processes (hydrolysis) of PifromATPdrivesmanycellular thesplitting

THEWORK OF CELLS

15

lntermediate f ilaments

Microtubules

FIGURE 1-15 The three types of cytoskeletalfilamentshave characteristic distributionswithin cells.Threeviewsof the same cell.A cultured fibroblast wastreatedwith threedifferent antibodv preparations. Eachantibody bindsspecifically to the protein monomers formingonetypeof filamentandischemically linkedto a differently colored fluorescent dye(green, blue,or red).Visualization

A n i m a l C e l l sP r o d u c eT h e i r O w n E x t e r n a l E n v i r o n m e nat n d G l u e s The simplest multicellular animals are sinsle cells embedded in a jelly of proteins and polysaccharides."ll.d th. extracellular matrix. Cells themselvesproduce and secretethesematerials, thus creating their own immediate environment (Chapter 19). Collagen, the single most abundant protein in the animal kingdom, is a major component of the extracellular matrix in most tissues.In animals, the extracellularmatrix cushions and lubricates cells. A specialized,especially tough matrix, the basal lamina, forms a supporting layer un_ derlying sheetlikecell layers and helps pr.u"rrt the cells from rlppmg apart. The cells in animal tissuesare ..glued" togerher by cell_ adhesionmolecules(CAMs) embeddedin their surfacemem_

Microfilaments

of the stainedcellin a fluorescence microscope reveals the location of filaments boundto a particular preparation dye-antibody In this case,intermediate filaments green;microtubules, arestained blue; and microfilaments, red.All threefibersystems contributeto the shapeandmovements of cells.lcourtesy of V Small ]

animal cells (Figure 1-15). The cytoskeleton prevents the plasma membrane of animal cells from relaxing into a sphere(Chapter 10); it also functions in cell locomotion and the intracellular transport of vesicles,chromosomes, and macromolecules(Chapters 17 and 18). The cytoskeletoncan be linked through the cell surfaceto the extracellular matrix or to the cytoskeletonof other cells,thus helping to form tissues(Chapter 19). All cytoskeletal filaments are long polymers of protein subunits. Elaborate systemsregulatethe assembly dirursembly of the cytoskeleton,thereby controlling cell".rd shape.In some cells the cytoskeletonis relatively stable, but in others it changesshapecontinuously. Shrinkageof the cytoskeleton in some parts of the cell and its growrh in other parts can produce coordinated changesin shape that result in cell locomotion. For instance,a cell can send out an extensionthat attachesto a surfaceor to other cellsand then retract the cell body from the other end. As this processcontinues due to coordinated changesin the cytoskeleton,the cell moves forward. Cells can move at rates on the order of 20 pm./second.

C e l l sC h a n g eS h a p ea n d M o v e

largement but not movement of cells from one position to another.

Cells changeshapeand move becausetheir internal skeleton. the cytoskeleton,exerts forces on the rest of the cell and its

CellsSenseand SendInformation

attachments- Three types of protein filaments, organized into networks and bundles, form the cytoskeleton within

A living cell continuously monitors its surroundings and adjusts its own activities and composition accordingly. Cells also communicate by deliberatelysendingsignalsthat can be receivedand interpreted by other cells. Suchsignalsare com_ mon not only within an individual organism but also be_ tween organisms.For instance,the odor of a pear signals a food source to us and other animals; consumption of the

16

.

c H A p r E1R | L I F E B E G t Nwst r H c E L L s

pear by an animal aids in distributing the pear'sseeds.Everyone benefits! The signals employed by cells include simple small chemicals, gases, proteins, light, and mechanical movements. Cells possessnumerous receptor proteins for detecting signals and elaborate pathways for transmitting them within the cell to evoke a response.At any time, a cell may be able to senseonly some of the signalsaround it, and how a cell responds to a signal may change with time. In some cases,receivingone signal primes a cell to respond to a subsequentdifferent signal in a particular way. Both changesin the environment (e.g.,an increaseor decreasein a particular nutrient or the light level) and signals receivedfrom other cells representexternal information that cells must process.The most rapid responsesto such signals generally involve changesin the location or activity of preexisting proteins. For instance, soon after you eat a carbohydrate-rich meal, glucosepours into your bloodstream.The rise in blood glucose is sensedby B cells in the pancreas, which respond by releasingtheir stored supply of the protein hormone insulin. The circulating insulin signal causesglucose transporters in the cytoplasm of fat and muscle cells to move to the cell surface,where they begin importing glucose. Meanwhile, liver cellsalso are furiously taking in glucosevia a different glucosetransporter.In both liver and musclecells, an intracellular signalingpathway triggeredby binding of insulin to cell-surfacereceptorsactivatesa key enzymeneeded to make glycogen,a large glucosepolymer (Figure 1-16a). The net result of thesecell responsesis that your blood glucoselevel falls and extra glucoseis stored as glycogen,which your cells can use as a glucosesource when you skip a meal to cram for a test. The ability of cells to send and respond to signalsis crucial to development. Many developmentallyimportant signals are secretedproteins produced by specific cells at specific times and places in a developing organism. Often a receiving cell integratesmultiple signals in deciding how to behave,for example, to differentiate into a particular tissue type, to extend a process,to die, to send back a confirming s i g n a l( y e s ,I ' m h e r e ! ) ,o r t o m i g r a t e . The functions of about half the proteins in humans, roundworms, yeast, and severalother eukaryotic organisms have beenpredicted basedon analysesof genomic sequences (Chapter6). Suchanalyseshave revealedthat at least10-15 percent of the proteins in eukaryotesfunction as secretedextracellular signals, signal receptors, or intracellular signaltransduction proteins, which pass along a signal through a seriesof steps culminating in a particular cellular response (e.g., increased glycogen synthesis).Clearly, signaling and signal transduction are maior activities of cells.

CellsRegulateTheir Gene Expressionto Meet C h a n g i n gN e e d s In addition to modulating the activities of existing proteins, cells often respond to changing circumstancesand to signals from other cells by altering the amount or types of proteins they contain. Gene expression,the overall processof selectively reading and using genetic information, is commonly

(a)

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lncreasedtranscription of specificgenes

1-16 Externalsignalscommonlycausea changein FIGURE the activity of preexistingproteinsor in the amountsand or of a hormone typesof proteinsthat cellsproduce.(a)Binding cantriggeran receptors to itsspecific molecule othersignaling of a the activity or decreases pathway that increases intracellular in to receptors bindingof insulin protein. Forexample, preexisting of cellsleadsto activation of liverandmuscle membrane the plasma from of glycogen in thesynthesis a keyenzyme glycogen synthase, within arelocated for steroidhormones glucose(b)Thereceptors complexes hormone-receptor The surface the cell cells,noton to increased tarqetgenes,leading of specific transcription activate that bindto proteins. Manysignals production of the encoded pathways, to alsoact,by morecomplex on the cellsurface receptors geneexPression modulate

controlled at the level of transcription, the first step in the production of proteins. In this way cells can produce a pariicular mRNA only when the encoded protein is needed' thus minimizing wasted energy. Producing an mRNA is, however,only the first in a chain of regulatedeventsthat together determine whether an active protein product is produced from a particular gene. Transcriptional control of geneexpressionwas first decisively demonstratedin the responseof the gut bacterium E. coli to different sugar sources.E. coli cells prefer glucose as a sugar source,but they can survive on lactosein a pinch' These bacteria use both a DNA-binding repressor protein and a DNA-binding actiuator protein to change the rate of transcription of three genesneededto metabolize lactosedepending on the relative amounts of glucoseand lactosepresint (Chapter 4). Such dual positiveinegativecontrol of gene exoression fine-tunes the bacterial cell's enzymatic equip-..rt fo. the job at hand. Like bacterial cells, unicellular eukaryotes may be subjected to widely varying environmental conditions that require extensivechangesin cellular structuresand function' T H EW O R K O F C E L L S

17

For instance,in starvation conditions yeastcellsstop growing and form dormant spores (seeFigure 1-6). In multicellular organisms, however, the environment around most cells is relatively constant. The major purpose of gene control in us and in other complex organismsis to tailor the properties of various cell types to the benefit of the entire animal or plant. Control of gene activity in eukaryotic cells usually involves a balance between the actions of transcriptional activators and repressors.Binding of activators to specificDNA regulatory sequencescalled enhancers turns on transcription, and binding of repressorsto other regulatory sequences called silencersturns off transcription. In Chapters 7 and 8, we take a closelook at transcriptional activators and repressors and how they operate, as well as other mechanismsfor controlling gene expression.In an extreme case,expression of a particular gene could occur only in part of the brain, only during eveninghours, only during a certain stageof development, only after a large meal, and so forth. Many external signals modify the activity of transcriptional activators and repressorsthat control specific genes. For example, lipid-soluble steroid hormones, such as estrogen and testosterone,can diffuse across the plasma membrane and bind to their specific receptors located in the cytoplasm or nucleus(Figure 1-16b).Hormone binding changes

Overview Animation: Life Cycle of a Cell {lltt Nondividing cells

o

Resting c el l s

R N Aa n d protein synthesis G2

A FIGURE 1-17 Duringgrowth, eukaryoticcellscontinually progressthrough the four stagesof the cell cycle, generatingnew daughtercells.In mostproliferating cells,the four phases of the cellcycleproceedsuccessively, takingfrom 10-20 hoursdepending on celltypeanddevelopmental state Duringinterphase, whichconsists of the G,,,S,andG2phases, the cellroughly doubles itsmassReplication of DNAdurinqthe S phaseleaves the cellwith four copiesof eachtypeof chromosome Inthe mitotic(M)phase, thechromosomes are evenlypartitioned to two daughter cells,andthecytoplasm divides roughlyin halfin mostcases. Undercertainconditions suchasstarvation or whena tissuehasreached itsfinalsize,cells willstopcycling andremainin a waitingstatecalledGs Mostcells in Gscanreenter thecycleif conditions chanqe.

18

C H A P T E R1

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L t F EB E G T N W S |TH CELLS

the shape of the receptor so that it can bind to specific enhancer sequencesin the DNA, thus turning the receptor into a transcriptional activator. By this rather simple signaltransduction pathway, steroid hormones cause cells to change which genes they transcribe (Chapter 7). Since steroid hormones can circulate in the bloodstream, they can affect the properties of many or all cells in a temporally coordinated manner. Binding of many other hormones and of growth factors to receptors on the cell surface triggers different signal-transduction pathways that also lead to changesin the transcription of specific genes (Chapters 15 and 16). Although thesepathways involve multiple components and are more complicated than those transducing steroid hormone signals,the generalidea is the same.

CellsGrow and Divide As we have discussed,reproduction is at the heart of biology; rocks don't do it. The reproduction of organisms depends on the reproduction of cells. The simplest type of reproduction entails the division of a "parent" cell into two "daughter" cells.This occurs as part of the cell cycle,a series of eventsthat preparesa cell to divide followed by the actual division process, called mitosis. The eukaryotic cell cycle commonly is representedas four stages(Figure 1-17). The chromosomes and the DNA they carry are copied during the S (synthesis)phase.The replicatedchromosomesseparare during the M (mitotic) phase,with each daughter cell getting a copy of each chromosome during cell division. The M and S phasesare separatedby two gap stages,the G1 phase and G2 phase, during which mRNAs and proteins are made. In single-celledorganisms,both daughtercellsoften (though not always) resemblethe parent cell. In multicellular organisms, stem cells can give rise to two different cells, one that resembles the parent cell and one that does not. Such asymmetric cell division is critical to the generationof different cell types in the body (Chapter21). During growth the cell cycle operatescontinuously with newly formed daughter cells immediately embarking on their own path ro mitosis. Under optimal conditions bacteria can divide to form two daughter cells once every 30 minutes. At this rate, in an hour one cell becomesfour: in a day one cell becomesmore than 101a, which if dried would weigh about 25 grams. Under normal circumstances, however, growth cannot continue at this rate becausethe food supply becomeslimiting. Most eukaryotic cells take considerably longer than bacterial cells to grow and divide. Moreover, the cell cycle in adult plants and animalsnormally is highly regulated(Chapter20). This tight control prevents imbalanced, excessivegrowth of tissueswhile ensuring that worn-out or damaged cells are replaced and that additional cells are formed in responseto new circumstancesor developmental needs.For instance, the proliferation of red blood cellsincreasessubstantiallywhen a person ascendsto a higher altitude and needs more capacity to capture oxygen. Somehighly specializedcells in adult animals, such as nervecellsand striatedmusclecells,rarely divide, if at all. The fundamental defect in cancer is loss of the ability to

control the growth and division of cells.In Chapter 25, we examine the molecular and cellular eventsthat lead to inappropriate, uncontrolled proliferation of cells. Mitosis is an asexual process since the daughter cells carry the exact samegeneticinformation as the parental cell. ln sexwalreproduction, fusion of two cells produces a third cell that contains genetic information from each parental cell. Sincesuch fusions would causean ever-increasingnumber of chromosomes, sexual reproductive cycles employ a special type of cell division, called meiosis,that reducesthe number of chromosomesin preparation for fusion (seeFigure 5-3). Cells with a full set of chromosomes are called diploid cells. During meiosis, a diploid cell replicates its chromosomes as usual for mitosis but then divides twice without copying the chromosomesin between. Each of the resulting four daughter cells, which have only half the full number of chromosomes,is said to be haploid. Sexual reproduction occurs in animals and plants and even in unicellular organismssuch as yeasts(seeFigure 1-5). Animals spendconsiderabletime and energygeneratingeggs and sperm, the haploid cells, called gametes,which are used for sexualreproduction. A human femalewill produce about half a million eggsin a lifetime, all of thesecells forming before she is born; a young human male produces about 100 million sperm each day. Gametes are formed from diploid precursor germ-line cells,which in humans contain 46 chromosomes.In humans the X and Y chromosomesare called sex chromosomes becausethey determine whether an individual is male or female. In human diploid cells, the 44 remaining chromosomes,called autosomes,occur as pairs of 22 different kinds. Through meiosis,a man produces sperm that have 22 chromosomes plus either an X or a Y, and a woman produces ova (unfertilized eggs) with 22 chromosomesplus an X. Fusion of an egg and sperm (fertilization) yields a fertilized egg,the zygote,with46 chromosomes,one pair of each of the 22 kinds and a pair of Xs in femalesor an X and a Y in males(Figure1-18). Errors during meiosiscan lead to disorders resulting from an abnormal number of chromosomes.These include Down's syndrome, caused by an extra chromosome 21, and Klinefelter's syndrome, causedby an extra X chromosome.

Diploid (2n)

Haploid(1nl

(",,o )

One type of female gamete

Two types of male gamete

Diploid (2n) Female zygote

Male zygote

meiosis 1-18 Dad madeyou a boy or girl. In animals, FIGURE Themale cellsformseggsandsperm(gametes). of diploidprecursor the sexof the parentproduces two typesof spermanddetermines sex chromosomes; Y are the here, X and asshown zygoteIn humans, fromthe maleparentto a Y chromosome the zygotemustreceive (non-sex chromosomes). developintoa male A : autosomes bing" between the fingers; the cells in the webbing subsequently die in an orderly and precisepattern that leavesthe fingers and thumb free to play the piano. Nerve cells in the brain soon die if they do not make proper or useful electrical

CellsDie from AggravatedAssaultor an lnternal Program 'lfhen

cells in multicellular organismsare badly damagedor infectedwith a virus, they die. Cell death resulting from such a traumatic event is messy and often releasespotentially toxic cell constituents that can damage surrounding cells. Cells also may die when they fail to receivea life-maintaining signal or when they receive a death signal. In this type of programmed cell death, called apoptosis, a dying cell actually produces proteins necessaryfor self-destruction.Death by apoptosis avoids the releaseof potentially toxic cell constituents(Figure1-19). Programmed cell death is critical to the proper development and functioning of our bodies (Chapter 21). During fetal life, for instance,our hands initially developwith "web-

1-19ApoPtoticcellsbreakapartwithout spewing FIGURE forth cell constituentsthat might harm neighboringcells. looklikethe cellon the left Cells Whitebloodcellsnormally likethe cellon the programmed celldeath(apoptosis), undergoing The arereleased blebsthateventually surface right,formnumerous is growth Apoptosis signals. it lackscertain cellisdyingbecause cellswherethey cells,to remove virus-infected to eliminate important (likethewebbingthatdisappears asfingersdevelop), arenot needed cellsthatwouldreactwith ourown immunesystem andto destroy Inc] Unlimited, Murtil/isuals bodies[Gopal THE WORK OF CELLS

19

connectionswith other cells. Some developinglymphocytes, the immune-systemcells intended to recognizeforeign proteins and polysaccharides,have the ability to reac against our own tissues.Such self-reactivelymphocytes becomeprogrammed to die before they fully marure. If these cells are not weededout before reaching maturity, they can causeautoimmune diseases,in which our immune system destroys the very tissuesit is meant to protect.

IE

InvestigatingCellsand TheirParts

To build an integrared understanding of how the various molecular components that underlie cellular functions work together in a living cell, we must draw on various perspectives. Here, we look at how five disciplines-cell biology, biochemistry and biophysics,genetics,genomics,and developmental biology-can conrribure to our knowledge of cell structure and function. The experimental approachesof each field probe the cell's inner workings in different ways, allowing us to ask different types of questions about cells and what they do. Cell division provides a good example to illustrate the role of different perspectivesin analyzing a complex cellular process.Although we discussthe different disciplinesseparatelyfor clarity, in practice most biologists use multiple approachesin concert. This is part of the fun of

Nanometers

The goal of cell biologists is to understandhow a cell is able to control its own shape and surface properties, transport materials to the right locations, copy itself, and receiveand send signals.Cell biologists use severaltypes of microscopy to observecells, while at the same time labeling specificcell components and altering them to seewhat happens.Analysesare generally done at the micrometer scale. Actual observation of cells awaited development of the first, crude microscopes in the early 1600s. A compound

Meters

Assemblies Macromolecules

Glucose

C-C bond

Cell Biology Revealsthe Size,Shape,Location, and Movementsof Cell Components

Millimeters

Micrometers

Small molecules Atoms

cell biologg putting together genetics with microscopy or enzymology with development. The realm of biology rangesin scalemore than a billionfold (Figure 1-20). Beyond that, it's ecology and earth science at the "macro" end, chemistry and physics at the "micro" end. The visible plants and animals that surround us are measuredin meters (100-102 m). By looking closely,we can seea biologicalworld of millimeters(1 mm : 10-3 m) and even tenths of millimeters (10-a m). Setting aside oddities like chicken eggs, most cells are 1-100 micrometers (1 pm : 10-5 m) long and thus clearlyvisibleonly when magnified. To seethe structureswithin cells, we must go farther down the sizescaleto 10-100 nanometers(1 nm : 10 v m).

Hemoglobin

M u l t i c e l l u l aor r g a n i s m s

Cells

Ribosome

C. elegans

Bacterium

Mitochondrion

Newborn human

Bumblebee

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10sm

1 0 ' 8m

'1 0-7m

0.1nm

1 nm

10nm

1 0 0n m

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1 0 - 5m 10pm

FIGURE 1-20 Biologistsare interestedin objectsrangingin sizefrom smallmolecules to the tallesttrees.A sampling of biological objects aligned on a logarithmic scale(a)TheDNAdouble helixhasa diameter of about2 nm (b)Eight-cell-stage numan embryothreedaysafterfertilization, about200 rr"macross(c)A wolf

20

CHAPTER 1

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L I F EB E G I N S WITHCELLS

1 0 - am 1 0 0u m

1 0 - 3m

1 0 2m

1 0 ' 1m

1 mm

1 0m m

100mm

1 0 0m 1m

spider, about15 mm across(d)Emperor penguins areabout1 m tall. [Part(a)Will andDeniMclntyrePart(b)YorgasNikas/photo Researchers, Inc Part(c)GaryGauglerl/isuals Unlimited, Inc Part(d)HughS Rosefuisuals Unlimited. lncl

microscope,the most useful type of light microscope,has two lenses.The total magnifying power is the product of the magnificationby each lens. As better lenseswere invented, the magnifying power and the ability to distinguish closely spacedobjects, the resolution, increasedgreatly. Modern cclmpoundmicroscopesmagnify the view about a thousandfold, so that a bacterium 1 micrometer (1 p-) long looks like it's a millimeterlong. Objectsabout 0.2 pm apart can be discernedin theseinstruments. Microscopy is most powerful when particular components of the cell are stained or labeled specifically,enabling them to be easilyseenand locatedwithin the cell. A simple exampleis stainingwith dyesthat bind specificallyto DNA to visualize the chromosomes. Specific proteins can be detected by harnessingthe binding specificityof antibodies,the proteins whose normal task is to help defend animals against infection and foreign substances.In general,eachtype of antibody binds to one protein or large polysaccharideand no other (Chapter 3). Purified antibodies can be chemically linked to a fluorescent molecule, which permits their detection in a specialfluorescencemicroscope(Chapter 3). lf a cell or tissueis treated with a detergentthat partially dissolvescell membranes,fluorescentantibodiescan drift in and bind to the specific protein they recognize. When the sample is viewed in the microscope,the bound fluorescent antibodies identify the location of the target protein (see F i g u r e1 - 1 5 ) . Betrer still is pinpointing proteins in living cells with intact membranes. One way of doing this is to introduce an engineeredgenethat codesfor a hybrid protein: part of the hybrid protein is the cellular protein of interest;the other part is a protein that fluoresceswhen struck by ultraviolet light. A common fluorescentprotein used for this purpose is green fluorescent protein (GFP), a natural protein that makes some iellyfish colorful and fluorescent. GFP "tagging" could reveal,for instance,that a particular protein is first made on the endoplasmic reticulum and then is moved by the cell into the lysosomes.In this case,first the endoplasmicreticulum and later the lysosomeswould glow in the dark. Chromosomesare visible in the light microscopeonly during mitosis,when they becomehighly condensed.The extraordinary behavior of chromosomesduring mitosis first was discoveredusing the improved compound microscopes of the late 1800s.About halfway through mitosis,the replicatedchromosomesbeginto move apart. Microtubules,one of the three types of cytoskeletalfilaments, participate in this movementof chromosomesduring mitosis.Fluorescenttagging of tubulin, the protein subunitthat polymerizesto form microtubules, reveals structural details of cell division that otherwisecould not be seenand allows observationof chromosomemovement(Figure1-21). Electron miqroscopesuse a focusedbeam of electronsinstead of a beam of light. In transmission electron microscopy, specimensare cut into very thin sections and placedunder a high vacuum,precludingexaminationof living cells. The resolution of transmission electron micro-

llll|

5e6nsAnimation:Mitosis

A FIGURE1-21 During the later stages of mitosis, microtubules (red) pull the replicated chromosomes(black) toward the ends of a dividing cell. Thisplantcell is stained and with a DNA-bindingdye (ethidium)to revealchromosomes antibodiesspecificfor tubulinto reveal with f luorescent-tagged microtubulesAt this stagein mitosis,the two copiesof each replicatedchromosome(calledchromatids)haveseparatedand are of AndrewBajer ] movinqawavf rom eachother [Courtesy

scopes,about 0.1 nm, permits fine structural details to be distinguished,and their powerful magnification would make a 1-pm-long bacterialcell look like a soccerball. Most of the organelles in eukaryotic cells and the double-layered structure of the plasma membrane were first observedwith electron microscopes(Chapter 9). \fith new specializedelectron microscopy techniques,three-dimensionalmodels of organellesand large protein complexes can be constructed from multiple images.But to obtain a more detailed look at the individual macromoleculeswithin cells, we must turn to techniqueswithin the purview of biochemistry and biophysics.

s e v e a tl h e B i o c h e m i s t r ay n d B i o p h y s i c R M o l e c u l a rS t r u c t u r ea n d C h e m i s t r yo f P u r i f i e d Cell Constituents Biochemistsextract the contentsof cellsand separatethe constituents based on differencesin their chemical or physical properties,a processcalledfractionation. The attention to individual moleculesmeans operating at the nanometer scale. Of particular interest are proteins, the workhorses of many A typical fractionation schemeinvolvesuse cellular processes. of various separationtechniquesin a sequentialfashion.These separationtechniquescommonly are basedon differencesin the size of moleculesor the electric charge on their surface (Chapter 3). To purify a particular protein of interest, a purification scheme is designed so that each step yields a

I N V E S T I G A T I NC GE L L SA N D T H E I RP A R T S

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FIGURE 1-22 Biochemicalpurificationof a protein from a cell extract often requiresseveralseparationtechniques.The purificationcanbe followedby gelelectrophoresis of thestarting proteinmixture andthefractions obtained fromeachpurification step Inthisprocedure, a sample isapplied to wellsin thetop of a gelatin-like slabandan electric fieldisappliedInthe presence of appropriate saltanddetergent concentrations, the proteins move throughthefibersof the geltowardtheanode,with largerproteins movingmoreslowlythroughthe gelthansmaller ones(seeFigure 3-35)Whenthegelisstained, proteins separated arevisible as distinctbandswhoseintensities areroughlyproportional to the proteinconcentration Shownhereareschematic depictions of oels for thestarting (lane1)andsamples mixture of proteins takenaiter eachof several purification stepsIn the firststep,saltf ractionation, proteinsthat precipitated with a certainamountof saltwereredissolved; electrophoresis (lane2) showsthatit of thissample contains fewerproteins thantheoriginal mixtureThesample then wassubjected in succession to threetypesof column chromatography proteins thatseparate by electrical charge, size,or bindingaffinityfor a particular (seeFigure smallmolecule 3-37).The finalpreparation isquitepure,ascanbe seenfromtheappearance of justoneproteinbandin lane5 [AfterJBergetal,2o02,Biochemistry W H Freeman andCompany, p 87l preparation with fewer and fewer contaminating proteins, until finally only the protein of interestremains (Figure 1-22). The initial purification of a protein of interest from a cell extract often is a tedious, time-consumingtask. Once a small amount of purified protein is obtained, antibodies to it can be produced by methods discussedin Chapter 19. For a biochemist, antibodies are near-perfecttools for isolating larger amounts of a protein of interest for further analysis.In effect, antibodies can "pluck out" the protein they specifically recognizeand bind from a semipure sample containing numerous different proteins. An increasinglycommon alterna-

22

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L I F EB E G I N SW I T H C E L L S

tive is to engineera gene that encodesa protein of interest with a small attached protein "tag," which can be used to pull out the protein from whole cell extracts. Purification of a protein is a necessaryprelude to studies on how it catalyzesa chemical reaction or carries out other functions and how its activiry is regulated. Some enzymesare made of multiple protein chains (subunits) with one chain catalyzing a chemical reaction and other chains regulating when and where that reaction occurs. The molecular machines that perform many critical cell processesconstitute even larger assembliesof proteins. By separating the individual proteins composing such assemblies,their individual catalytic or other activitiescan be assessed. For example,purification and study of the activity of the individual proteins composing the DNA replication machine provided clues about how they work together to replicate DNA during cell division (Chapter 4). The folded, three-dimensionalstructure, or conformation, of a protein is vital to its function. To understand the relation between the function of a protein and its form, we need to know both what it does and its detailed structure. The most widely used method for determining the complex structures of proteins, DNA, and RNA is x-ray crystallography, one of the powerful tools of biophysics.Computer-assistedanalysisof the data often permits the location of every atom in a large, complex molecule to be determined. The double-helix structure of DNA, which is key to its role in herediry was first proposed basedon x-ray crystallographic studies.Throughout this book you will encounter numerous examplesof protein structuresas we zero in on how proteins work.

GeneticsRevealsthe Consequences of DamagedGenes Biochemical and crystallographic studies can tell us much about an individual protein, but they cannot prove that it is required for cell division or any other cell process.The importance of a protein is demonstratedmost firmly if a mutation that prevents its synthesis or makes it nonfunctional adverselyaffects the processunder study. !(/e define the genotype of an organism as its composition of genes;the term also is commonly used in referenceto different versionsof a singlegeneor a small number of genes of interest in an individual organism. A diploid organism generally carries two versions (alleles)of each gene, one derived from each parent. There are important exceptions, such as the geneson the X and Y chromosomesin males of some species,including our own. The phenotype is the visible outcome of a gene's action, such as blue eyes versus brown eyesor the shapesof peas.In the early days of genetics, the location and chemical identity of genes were unknown; only the observablecharacteristics,the phenotypes, could be followed. The concept that genesare like "beads" on a long "string," the chromosome,was p,roposedearly in the 1900s basedon geneticwork with the fruit fly Drosophila. In the classicalgeneticsapproach, mutants are isolated that lack the ability to do something a normal organism can do. Often large genetic "screens" are done to look for many different mutant individuals (e.g., fruit flies, yeast cells) that are

unable to complete a certain process,such as cell division or muscle formation. In experimental organismsor cultured cells, mutations usually are produced by treatment with a mutagen, a chemical or physical agent that promotes mutations in a largely random fashion.But how can we isolateand maintain mutant organismsor cellsthat are defectivein some process, such as cell division, that is necessaryfor survival?One way is to look for organisms with a temperature-sensitivemutation. Thesemutants are able to grow at one temperature,the permissiue temperature, but not at anothe5 usually higher temperature, the nonpermissiuetemperature.Normal cells can grow at either temperature. In most cases,a temperature-sensitivemutant produces an altered protein that works at the permissive temperaturebut unfolds and is nonfunctional at the nonpermissive temperature. Temperature-sensitive screensare readily done with viruses,bacteria, yeast,roundworms, and fruit flies. By analyzing the effectsof numerous different temperaturesensitivemutations that altered cell division, geneticistsdiscoveredall the genesnecessaryfor cell division without knowing anything, initially, about which proteins they encode or how theseproteinsparticipatein the process.The greatpower of geneticsis to reveal the existenceand relevanceof proteins without prior knowledge of their biochemicalidentity or molecular function. Eventually these "mutation-defined" genes were isolatedand replicated(cloned)with recombinantDNA techniquesdiscussedin Chapter 5. \fith the isolatedgenesin hand, the encodedproteins could be produced in the test tube or in engineeredbacteria or cultured cells. Then the biochemistscould investigatewhether the proteins associatewith other proteins or DNA or catalyze particular chemical reactions during cell division (Chapter20). The analysis of genome sequencesfrom various organisms during the past decadehas identified many previously unknown DNA regions that are likely to encode proteins (i.e., protein-coding genes).The generalfunction of the protein encoded by a sequence-identifiedgene may be deduced by analogy with known proteins of similar sequence.Rather than randomly isolating mutations in novel genes, several techniquesare now available for inactivating specific genes by engineering mutations into them or destroying their mRNA with interfering RNA molecules(Chapter 5). The effects of such deliberategene-specificinactivation procedures provide information about the role of the encoded proteins in living organisms. This application of genetic techniques starts with a gene/proteinsequenceand ends up with a mutant phenotype; traditional genetics starts with a mutant phenotype and ends up with a gene/proteinsequence.

GenomicsRevealsDifferencesin the Structure and Expressionof EntireGenomes Biochemistry and geneticsgenerally focus on one gene and '$flhile powerful, these tradiits encoded protein at a time. tional approachesdo not give a comprehensiveview of the structure and activity of an organism'sgenome,its entire set of genes.The field of genomicsdoes just that, encompassing the molecular characterizationof whole genomesand the determination of global patterns of geneexpression.The recent

completion of the genome sequencesfor more than 100 speciesof bacteria and severaleukaryotesnow permits comparisons of entire genomes from different species.The results provide overwhelming evidenceof the molecular unity of life and the evolutionary processesthat made us what we are (seeSection 1.5). Genomics-basedmethods for comparing thousands of piecesof DNA from different individuals all at the same time are proving useful in tracing the history and migrations of plants and animals and in following the inheritance of diseasesin human families. DNA microarrays can simultaneously detect all the mRNAs presentin a cell, thereby indicating which genesare being transcribed. Such global patterns of gene expression clearly show that liver cells transcribe a quite different set of genesthan do white blood cellsor skin cells. Changesin gene expressionalso can be monitored during a diseaseprocess' in responseto drugs or other external signals'and during development. For instance,the identification of all the mRNAs presentin cultured fibroblasts before, during' and after they divide has given us an overall view of transcriptional changesthat occur during cell division (Figure 1-23). Cancet diagnosis is being transformed becausepreviously indistinguishablecancer cells have distinct gene expressionpatterns and prognoses (Chapter 25). Similar studies with different organisms and cell types are revealing what is universal about the genesinvolved in cell division and what is specific to particular organisms.To find out which genesare directly regulatedby a transcription factor, chromatin containing the protein of interest can be purified with an antibody and the associated DNA analyzed on microarrays, a procedure called chromatin immunopreclpltatlon. The entire complement of proteins in a cell, its proteome' is controlled in part by changesin genetranscription.The regulated synthesis,processing,localization' and degradationof specific proteins also play roles in determining the proteome of a particular cell. Learning how proteins bind to other proteins, often in large, multiprotein complexes, is providing a comprehensiveview of the molecular machines important for cell functioning. The field of proteomics will advance dramatically once high-throughput x-ray crystallography, currently under development,permits researchersto rapidly determine the structuresof hundredsor thousandsof proteins.

DevelopmentalBiology RevealsChangesin the Propertiesof Cellsas They Specialize Another approach to viewing cells comes from studying how they changeduring development of a complex organism. Bacteria, algae, and unicellular eukaryotes (protozoans, yeasts) often, but by no means always, can work solo. The concerted actions of the trillions of cells that compose our bodies require an enormous amount of communication and division of labor. During the development of multicellular organisms, differentiation processesform hundreds of cell types, each specialized for a particular task: transmission of electric signals by neurons, transport of oxygen by red blood cells, destruction of infecting bacteria by macrophages' contraction by muscle cells, chemicalprocessingby liver cells,and so on. I N V E S T I G A T I NC GE L L SA N D T H E I RP A R T S

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EXPERIMENTAL FIGURE 1-23 Microarrayanalysisof normal growing braincellsand braintumor cells.An experiment likethis isa starting pointfor learning howtumorcellsdifferfromnormal cellsRNAwasextracted fromnormalgrowingmousebraincells fromthecerebellum andfroma tumorof thecerebellum TheRNA fromthe tumorwaslabeled with a reddye,andRNAfromthe normal,non-tumorous cerebellum waslabeled with a greendye The two RNApreparations weremixedandhybridized to a microarray containing thousands of spotsof DNA Eachspotcontains the DNA sequence of onegene Unbound RNAwaswashedawayandthe microarray wasexposed to UVlight,whichcauses the dyesto fluoresce Spotsthataregreenhaveboundmostlynormalcerebellum RNA,spotsthatareredhaveboundmostlytumorRNA,andspots thatareyellowhaveboundroughly equalamounts of each.The faintlystained spotsrepresent genesfor whichthereislittleRNAin eithersampleThedataindicate whichgeneshavebeentranscribed in tumors,normalcerebellum, or both Onlypartof the datais shownhereTheentiredatasetrequires analyzing the colorsof more than25,000spots,allof whichcanbefittedontoonemicroscope slide.Precise measurements of colorintensity areactually madeby a spectrophotometer, but lookingby eyeshowsthat manygenesare morehighlyexpressed in normalor tumorcells.Someof these differences arethe consequence of the changeintotumorcells,but somemayreveal geneexpression changes thatcausethetumorsto form In addition, proteins madeexclusively in tumors,andperhaps necessary for uncontrolled growth,maybe candidate targets for discovering anti-cancer drugs [Courtesy of TalRaveh andl\y'atthew Scott, StanfordUniversitySchoolof Medicinel

Many of the differences among differentiated cells are due to production of specificsetsof proteins neededto carry out the unique functions of each cell type; that is, only a subsetof an organism'sgenesis transcribedat any given time or in any given cell. Such differential gene expressionat different times or in different cell types occurs in bacteria, fungi, plants, animals, and even viruses. Differential gene

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expression is readily apparent in an early fly embryo, in which all the cells look alike until they are stained to detect the proteins encoded by particular genes (Figure 1-24). Transcription can changewithin one cell type in responseto an external signal or in accordancewith a biological clock; some genes,for instance,undergo a dalIy cycle between low and high transcription rates. Producing different kinds of cells is not enough to make an organism, any more than collecting all the parts of a truck in one pile gives you a truck. The various cell types must be organized and assembled into all the tissues and organs. Even more remarkable, these body parts must work almost immediately after their formation and continue working during the growth process. For instance, the human heart begins to beat when it is lessthan 3 mm long, when we are mere 23-day-old embryos, and continues beating as it grows into a fist-sizemuscle. From a few hundred cells to billions, and still ticking. In the developing organism, cells grow and divide at some times and not others, they assembleand communicate, they prevent or repair errors in the developmentalprocess, and they coordinate each tissuewith others. In the adult organism,cell division largely stops in most organs.If part of an organ such as the liver is damaged or removed, cell division resumesuntil the organ is regenerated.The legend goes that Zets punished Prometheus for giving humans fire by

FIGURE 1-24 Differentialgene expressioncan be detectedin early fly embryosbefore cellsare morphologicallydifferent. An earlyDrosophila embryohasabout6000cellscoveringitssurface, mostof whichareindistinguishable by simplelightmicroscopy. lf the embryois madepermeable to antibodies with a detergent that partially dissolves membranes, theantibodies canfindandbindto the proteins theyrecognize Inthisembryowe seeantibodies tagged with a fluorescent labelboundto proteins thatarein the nuclei; each smallsphere corresponds to onenucleus. Threedifferent antibodies wereused,eachspecific proteinandeachgivinga for a different distinct green,or blue)in a fluorescence color(yellow, microscope. Theredcolorisaddedto highlight overlaps between theyellowand bluestainsThelocations proteins of thedifferent showthatthe cells arein factdifferent at thisearlystage,with particular genesturned on in specific stripes of cells. Thesegenescontrolthesubdivision of the bodyintorepeating segments, likethe blackandyellowstripes of a hornet.[Courtesy of Sean Carroll, University of Wisconsin ]

chaining him to a rock and having an eagleeat his liver. The punishment was eternal because,as the Greeks evidently knew, the liver regenerates. Developmental studies involve watching where, when, and how different kinds of cells form, discovering which signals trigger and coordinate developmentalevents,and understandingthe differential gene action that underliesdifferentiation (Chapters 16 and 21). During development we can see cells change in their normal context of other cells. Cell biology, biochemistry, cell biology, genetics, and genomics approachesare all employed in studying cells during development.

C h o o s i n gt h e R i g h t E x p e r i m e n t aOl r g a n i s m for the Job Our current understanding of the molecular functioning of cells rests on studieswith viruses,bacteria, yeast, protozoa, slime molds, plants, frogs, sea urchins, worms, insects,fish, chickens, mice, and humans. For various reasons, some organisms are more appropriate than others for answering particular questions.Becauseof the evolutionary conservation of genes,proteins, organelles,cell types, and so forth, discoveriesabout biological structuresand functions obtained with one experimental organism often apply to others. Thus researchersgenerallyconduct studieswith the organism that is most suitable for rapidly and completely answering the question being posed, knowing that the results obtained in one organismare likely to be broadly applicable.Figure1-25 summarizesthe typical experimental usesof various organisms whose genomeshave been sequencedcompletely or nearly so. The availability of the genomesequencesfor these organisms makes them particularly useful for geneticsand genomicsstudies. Bacteria have several advantagesas experimental organisms: they grow rapidlS possesselegant mechanisms for controlling gene activity, and have powerful genetics. This latter property relates to the small size of bacterial genomes,the easeof obtaining mutants, the availability of techniquesfor transferring genes into bacteria, an enormous wealth of knowledge about bacterial gene control and protein functions, and the relative simplicity of mapping genesrelative to one another in the genome. Singlecelledyeastsnot only have some of the sameadvantagesas bacteria but also possessthe cell organization,marked by the presenceof a nucleusand organelles,that is characteristic of all eukaryotes. Studiesof cellsin specializedtissuesmake use of animal and plant "models," that is, experimentalorganismswith attributes typical of many others. Nerve cells and muscle cells, for instance,traditionally were studied in mammals cells,such or in creatureswith especiallylarge or accessible as the giant neural cells of the squid and sea hare or the flight musclesof birds. More recently,muscle and nerve development have been extensively studied in fruit flies (Drosophila melanogaster),roundworms (Caenorhabditis elegans),and zebrafish(Danio rerio), in which mutants can be readily isolated. Organisms with large-celledembryos

that develop outside the mother (e.g.' frogs, sea urchins, fish, and chickens) are extremely useful for tracing the fates of cells as they form different tissues and for making extracts for biochemicalstudies.For instance,a key protein in regulating mitosis was first identified in studies with frog and sea urchin embryos and subsequentlypurified from extracts (Chapter 20lt. Using recombinant DNA techniques,researcherscan engineer specificgenesto contain mutations that inactivate or increaseproduction of their encodedproteins. Suchgenescan be introduced into the embryos of worms, flies, frogs, sea urchins, chickens,mice, a variety of plants, and other organisms,permitting the effectsof activating a geneabnormally or inhibiting a normal gene function to be assessed.This approach is being used extensivelyto produce mouse versions of human genetic diseases.Inactivating particular genes by introducing short piecesof interfering RNA is allowing quick tests of gene functions possiblein many organisms.The expansion of genomeproiectsto critically important diseaseorganisms,such as malaria, and to creaturesthat span the evolutionary tree is bringing new options for medicine and new insights into how living organisms have diversified to take advantageof every possibleecologicalniche. Mice have one enormous advantage over other experimental organisms:they are the closestto humans of any animal for which powerful geneticapproachesare feasible.Engineered mouse genes carrying mutations similar to those associatedwith a particular inherited diseasein humans can be introduced into mouse embryonic stem (ES) cells. These cells can be injected into an early embryo, which is then implanted into a pseudopregnantfemale mouse (a mouse treated with hormones to trigger physiological changes neededfor pregnancy) (Chapter 5). If the mice that develop from the injected ES cells exhibit diseasessimilar to the human disease,then the link between the diseaseand mutations in a particular geneor genesis supported. Once mouse models of a human diseaseare available, further studies on the molecular defectscausing the diseasecan be done and new treatmentscan be tested,thereby minimizing human exposure to untested treatments. Large-scalegenetic screens are being done that take advantageof newly designedmutagenic transposons.The transposons allow efficient generation of mouse mutants and rapid identification of the gene that has been hit in each one. A continuous unplanned genetic screen has been per\What we mean formed on human populations for millennia. is that all sorts of human variations have arisen and have been noticed, since they affect visible or noticeable human characteristics.Thousandsof inherited traits have beenidentified and, more recently,mapped to locations on the chromosomes.Some of these traits are inherited propensitiesto get a disease;others are eye color or other minor characteristics. Geneticvariations in virtually every aspectof cell biology can be found in human populations, allowing studiesof normal and diseasestatesand of variant cells in culture. Less-commonexperimental organisms offer possibilities for exploring unique or exotic properties of cells and for studying standard properties of cells that are exaggeratedin I N V E S T I G A T I NC GE L L SA N D T H E I RP A R T S

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Podcast:Common ExperimentalOrganisms

Viruses

Bacteria

Proteinsinvolvedin DNA, RNA, protein synthesis Gene regulation Cancerand control of cell oroliferation Transportof proteinsand organellesinsidecells Infectionand immunity Possiblegene therapy approaches

Proteinsinvolvedin DNA, RNA, protein synthesis, metabolism Gene regulation Targetsfor new antibiotics Cell cycle Signaling

Yeast (Saccharo myce s cerev is i ael

Roundworm I Caenorh abd itis elegansl

Controlof cell cycle and cell division Proteinsecretionand membrane biogenesis Functionof the cytoskeleton Cell differentiation Aging Gene regulationand chromosome structure

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Developmentof the body plan C e l ll i n e a g e Formationand function of the nervous system Controlof programmedcell death Cell proliferationand cancergenes Aging Behavior Gene regulationand chromosome structure

Fruit ffy (Drosophi Ia mel anoga sterl

Zebrafish

Developmentof the body plan Generationof differentiatedcell lineages Formationof the nervoussystem, heart,and musculature Programmedcell death Geneticcontrol of behavior Cancergenesand control of cell proliferation Controlof cell polarization Effectsof drugs, alcohol,pesticides

Developmentof vertebratebody tissues Formationand function of brain and nervous syslem Birth defects Cancer

Mice, includingculturedcells

Plant (A ra b i d ops is th aIi anal

Developmentof body tissues F u n c t i o no f m a m m a l i a ni m m u n e system Formationand function of brain and nervoussystem Models of cancersand other human diseases Gene regulationand inheritance Infectiousdisease

Developmentand patterningof tissues Geneticsof cell biology Agricultural applications Physiology Gene regulation lmmunity Infectiousdisease

L I F EB E G I N SW I T H C E L L S

< FIGURE 1-25 Eachexperimentalorganismusedin cell (a) biology has advantagesfor certaintypes of studies.Viruses (b)havesmallgenomes andbacteria amenable to genetic dissection Manyinsights intogenecontrolinitially camefromstudies with these (c)hasthe cellular organismsTheyeastSaccharomyces cerevisiae organization of a eukaryote but isa relatively simplesingle-celled genetically. organism thatiseasyto growandto manipulate Inthe (d),whichhasa small nematode worm Caenorhabditis elegans numberof cellsarranged in a nearlyidentical wayin everyworm,the formation of eachindividual cellcanbe tracedThefruitfly (e),firstusedto discover Drosophila melanogaster the properties of genesthat chromosomes, hasbeenespecially valuable in identifying controlembryonic development Manyof thesegenesare evolutionarily conserved in humansThezebraf ishDaniorerio(f)is genesthatcontrol usedfor rapidgenetic screens to identify development andorganogenesis. Of theexperimental animal (g)areevolutionarily systems, mice(Musmusculus) the closestto humans andhaveprovided models for studying numerous human genetic andinfectious diseases. Themustard-family weed Arabidopsis thaliana,sometimes described asthe Drosophila of the plantkingdom, genes hasbeenusedfor genetic screens to identify involved in nearlyeveryaspect of plantlife Genome sequencing is completed for manyviruses andbacterial species, theyeast Saccharomyces cerevisiae, the roundwormC. elegans,the f ruit fly D. melanogaste4 humans,andthe plantArabidopsis thalianallis mostlycompleted for miceandin progress for zebrafish Other particularly organisms, frogs,seaurchins, chickens, andslimemolds, to be immensely continue valuable for cellbiologyresearch Increasingly, a widevariety of otherspecies for areused,especially (a)Visuals studies of evolution of cellsandmechanisms Unlimited, [Part Inc Part(b)KariLountmaa/Science Photo Library/Photo Researchers, Inc Part (c)ScimavPhoto Researchers, Inc Part(d)Photo Researchers, IncPart(e) Darwin Dale/Photo Researchers, Inc,Part(f)IngeSpencel'/isuals Inc Unlimited, Part(g)J M Labavjancanatuisuals Unlimited, Inc Part(h)Darwin Dale/Photo Researchers, Inc]

a useful fashion in a particular animal. For example, the ends of chromosomes,the telomeres,are extremely dilute in most cells.Human cells typically contain 92 telomeres(46 chromosomesX 2 endsper chromosome).In contrast,some protozoawith unusual "fragmented" chromosomescontain millions of telomeres per cell. Taking advantage of the unique properties of this well-chosen experimental organism has led to important recent discoveriesabout telomere structure.

The Most SuccessfulBiologicalStudiesUse M u l t i p l eA p p r o a c h e s We have discussedfive classesof approachesto biological problems: cell biology biochemistry and biophysics, genetics, genomics,and developmentalbiology. Each has its own types of experiments,and most biological problems require more than one approach in order to reach a satisfying understanding of mechanism. Now we will survey how these approacheshave been applied to the study of cell division to emphasizehow important it is to use multiple types of experlments.

Cell division was viewed, and indeed discovered,by some of the earliest usersof microscopes.More recently a variety of kinds of microscopy, including confocal and electron microscopy and time-lapseimaging (Chapter 9), have been used to characterizethe stepsof the cell cycle. Most biology begins with this sort of observation, defining the mysteries that must be tackled experimentally.Then manipulations begin. Antibodies were made againstproteins that play critical roles in cell division both to detect proteins and in some casesto interfere with the functions of those proteins. Key proteins were fused to fluorescentproteins, starting with the iellyfish greenfluorescentprotein (GFP),so that key proteins could be followed in living cells. Questions about when and where proteins work could be addressedand their functions defined to an extent. The apparatusof cell division, such as the mitotic spindle and other protein complexes,was purified and analyzed using the approaches of biochemistry and biophysics. Each protein had to be purified to find out whether it is part of a complex of proteins bound together as a machine, and the structuresof key proteins were determined using x-ray crystallography and other methods (Chapter 3). Previously unknown enzymatic activities were detected in extracts using assays such as measuring the attachment of phosphate groups to cell division regulatory proteins by kinases, and then the relevant kinasescould be purified. Finding a novel protein in a complex of proteins involved in cell division makes it a good bet that the protein does something important, a sort of "guilt by association," but it doesnot provide proof that the protein matters. For that one must turn to genetics. Genetics can be used to identify mutants in the newly found protein. If cell division fails in a living organism when the protein is not working, you know the protein matters. Geneticsis also a way to identify previously unknown genesand proteins sincescreenscan be done (especiallyin bacteria and yeast, but also in more complex Iab organisms)to look for all the genesthat are neededfor cell division. The newly discoveredproteins can be incorporated into a complete picture of the mechanicsof cell division machinery. Genomics provides another way to look for working parts of the cell-division machine. Since it is often true that mRNAs and proteins are produced only when they are needed, using microarrays to look for all geneswhose expressionvaries with the cell cycle is a powerful approach to identify candidatesfor cell division regulators. Having identified new genesthat are required for cell division, one must find out how thesegenes'protein products work. Simply knowing that a protein matters to cell division is not enough to understand the mechanism.Thus it is necessaryto return to biochemical and biophysical approaches to work out the molecular biology and to cell biology to monitor protein locations and movements. Finally, cell division does not happen in a vacuum; it happens in the context of the life cycle of the organism. To fully appreciatehow the regulation works and is used, it is important to use the approachesof developmental biology

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to revealwhen and where cell division normally happensand why. In theseexperimentsthe times and placesof cell division during the developmentof the organism are monitored, and then the signals that stimulate or suppresscell division are identified and studied. Errors in developmental control of cell division, revealedby studying mutants, can causeorgans and tissuesto be the wrong sizeor cancer. The samekinds of approachesthat work for cell division can be applied to many other biological challenges,such as Iearning how muscles form or how they work or how the brain functions. Often it's good to use every tool in the toolkit.

tf,|

A GenomePerspective

on Evolution Comprehensivestudies of genesand proteins from many organismsare giving us an extraordinary documentationof the history of life. Nature is a laboratory that has been conducting experimentsfor billions of years, and some of the most successfulgenomesthat emergedare still with us. 'We share with other eukaryotes thousands of individual proteins, hundredsof macromolecularmachines,and most of our organelles,all as a result of our sharedevolutionary history. New insights into molecular cell biology arising from genomicsare leadingto a fuller appreciationof rhe elegant molecular machines that arose during billions of yearsof genetictinkering and evolutionaryselectionfor the most efficient, precise designs. Due ro alternative RNA splicing,the number of proteins vastly exceedsthe number of genes,and the functions of many variant proteins and assembliesof proteins remain to be discovered. Once a more complete description of cells is in hand, we will be ready to fully investigate the rippling, flowing dynamics of living systems.

MetabolicProteins,the GeneticCode, a n d O r g a n e l l eS t r u c t u r e sA r e N e a r l yU n i v e r s a l Even organisms that look incredibly different share many biochemical properties. For instance, the enzymesthat catalyze degradation of sugars and many other simple chemical reactions in cells have similar structures and mechanisms in most living things. The genetic code whereby the nucleotide sequencesof mRNA specifiesthe amino acid sequencesof proteins can be read equally well by a bacterial cell and a human cell. Becauseof the universalnature of the genetic code, bacterial "factories" can be designedto manufacture growth factors, insulin, clotting factors, and other human proteins with therapeutic uses. The biochemical similarities among organisms also extend to the organelles found in eukaryotic cells.The basic structuresand functions of these subcellular components are largely conservedin all eukaryotes.

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Computer analysisof DNA sequencedata, now available for numerousbacterialspeciesand severaleukaryotes, can locate protein-coding geneswithin genomes.\fith the aid of the geneticcode, the amino acid sequencesof proteins can be deduced from the corresponding gene sequences.Although simple conceptually, "finding" genes and deducing the amino acid sequencesof their encoded proteins is complicated in practice becauseof the many noncodingregionsin eukaryoticDNA (Chapter5). Despite the difficulties and occasional ambiguities in analyzing DNA sequences, comparisonsof the genomesfrom a wide range of organisms provide stunning, compelling evidence for the conservation of the molecular mechanisms that build and change organisms and for the common evolut i o n a r y h i s t o r yo f a l l s p e c i e s .

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Darwin'sldeasAbout the Evolutionof Whole A n i m a l sA r e R e l e v a n t o G e n e s Darwin did not know that genesexist or how they change, but we do: the DNA replication machine makes an error, or a mutagen causesreplacement of one nucleotide with another or breakage of a chromosome. Some changesin the genomeare innocuous, some mildly harmful, some deadly; a very few are beneficial. Mutations can change the sequence of a gene in a way that modifies the activity of the encoded protein or alters when, where, and in what amounts the protein is produced in the body. Gene-sequencechangesthat are harmful will be lost from a population of organisms becausethe affected individuals cannot survive as well as their relatives. This selection process is exactly what Darwin described without knowing the underlying mechanismsthat cause organisms to vary. Thus the selectionof whole organisms for survival is really a selectionof genes,or more accurately sets of genes.A population of organismsoften contains many variants that are all roughly equally well-suited to the prevailing conditions. When conditions change-a fire, a flood, loss of preferred food supply, climate shift-variants that are better able to adapt will survive, and those less suited to the new conditions will begin to die out. In this way, rhe genetic composition of a population of organisms can change over tlme.

M a n y G e n e sC o n t r o l l i n gD e v e l o p m e n t A r e R e m a r k a b l yS i m i l a ri n H u m a n s a n d O t h e rA n i m a l s As humans, we probably have a biasedand somewhat exaggerated view of our status in the animal kingdom. Pride in our swollen forebrain and its associatedmental capabilities may blind us to the remarkably sophisticated abilities of other species:navigation by birds, the sonar systemof bats, homing by salmon, or the flight of a fIy. Despite all the evidencefor evolutionary unity at the cellular and physiological levels,everyoneexpectedthat genes

(a)

Genes

Flv

(d)

Mammal

(e)

< FIGURE 1-26 Similargenes,conservedduringevolution, processes in diverseanimals. regulatemanydevelopmental to havehada commonancestor areestimated Insects andmammals abouthalfa billionyearsago.Theysharegenesthatcontrolsimilar of the processes, suchasgrowthof heartandeyesandorganization times. conservation of functionfromancient bodyplan,indicating (a)Hoxgenesarefoundin clusters of mostor on thechromosomes proteins thatcontrolthe allanimalsHoxgenesencoderelated of activities of othergenesHoxgenesdirectthedevelopment as axisof manyanimals, alongthe head-to-tail different segments Eachgeneisactivated colors. indicated by corresponding (transcriptionally) axisand regionalongthe head-to-tail in a specific in micethe Hox there Forexample, controls the growthof tissues of vertebrae shapes genesareresponsible for thedistinctive Hoxgenesin fliescausebodypartsto formin affecting Mutations on the head. suchaslegsin lieuof antennae thewronglocations, genesprovide address andserveto direct a head-to-tail These in the rightplaces(b)Development of the rightstructures formation a genecalled eyesin fruitfliesrequires of the largecompound (c)Flieswith inactivated (namedfor the mutantphenotype). eyeless the human geneslackeyes(d)Normalhumaneyesrequire eyeless (e)Peoplelacking to eyeiess. gene,calledPax6,that corresponds anrrdia,a lackof adequatePax6functionhavethe geneticdisease encodehighlyrelatedproteins irisesin the eyesPax6andeyeless from of othergenesandaredescended theactivities that regulate (b)and(c)Andreas gene lParts Hefti, Interdepartmental the sameancestral (lEM) of BaselPart(d) of theUniversity Biocenter Microscopy Electron Unlimitedl Inc Part(e)Visuals Researchers. Fraser/Photo @Simon

that, as far as we can tell, are utterly absentfrom certain lineagesof animals. Plants, not surprisingly,exhibit many such differencesfrom animals after a billion-year separation in their evolution. Yet certain DNA-binding proteins differ between peasand cows at only two amino acids out of 1'021

regulating animal development would differ greatly from one phylum to the next. After all, insectsand seaurchins and mammals look so different. \(e must have many unique proteins to createa brain like ours . . . or must we? The fruits of research in developmental genetics during the past two decadesrevealthat insectsand mammals, which have a common ancestor about half a billion years ago, possessmany genes(Figure 1-26). Indeed, similar development-regulating a large number of these genes appear to be conserved in many and perhaps all animals. Remarkably, the developmental functions of the proteins encoded by thesegenesare also often preserved.For instance,certain proteins involved in eye development in insects are related to protein regulators of eye developmentin mammals. Samefor development of the heart, gut, lungs, and capillaries and for placementof body parts along the head-to-tail and back-to-front body axes (Chapter19). This is not to say that all genesor proteins are evolutionarily conserved. Many striking examples exist of proteins

H u m a nM e d i c i n el s I n f o r m e db y R e s e a r c h on OtherOrganisms Mutations that occur in certain genesduring the course of our lives contribute to formation of various human cancers. The normal, wild-type forms of such "cancer-causing"genes generallyencodeproteins that help regulatecell proliferation or death (Chapter 21').'Wealso can inherit from our parents mutant copies of genesthat causeall manner of genetic diseases,such as cystic fibrosis, muscular dystrophy' sickle cell anemia, and Huntington's disease.Happily we can also inherit genesthat make us robustly resist disease.A remarkable number of genesassociatedwith cancer and other human diseasesare present in evolutionarily distant animals. For example, a recent study shows that more than threequarters of the known human diseasegenes are related to genesfound in the fruit fIy Drosophila. Vith the identification of human diseasegenesin other organisms,experimental studies in experimentally tractable organisms should lead to rapid progress in understanding

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the normal functions of the disease-relatedgenes and what occurs when things go awry. ConverselS the disease states themselvesconstitute a genetic analysis with well-studied phenotypes. All the genesthat can be altered to cause a certain diseasemay encode a group of functionally related proteins. Thus clues about the normal cellular functions of pro-

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teins come from human diseasesand can be used to guide initial research into mechanism. For instance, genesinitially identified becauseof their link to cancer in humans can be studied in the context of normal development in various model organisms, providing further insight about the functions of their protein products.

CHAPTER

CHEMICAL FOUNDATIONS Polarized light microscopic imageof crystalsof ATBwhose hydrolysis isa primarysourceof energythat drivesmanycellular chemical reactions[Dr.ArthurM SiegelmaMr'isuals Unlimited ]

he life of a cell dependson thousands of chemical interactions and reactions exquisitely coordinated with one another in time and sDaceand under the influence of the cell's genetic instructions and its environment. By understandingat a molecular level theseinteractions and reactions, we can begin to answer fundamental questionsabout cellular life: How doesa cell extract critical nutrients and information from its environment? How does a cell convert the energy stored in nutrients into work (movement,synthesis of critical components)?How does a cell transform nutrients into the fundamental structuresrequired for its survival (cell wall, nucleus, nucleic acids, proteins, cytoskeleton)? How does a cell link itself to other cells to form a tissue? How do cells communicate with one another so that a complex, efficiently functioning organism can develop and thrive? One of the goals of Molecular Cell Biology is to provide answers to these and other questions about the structure and function of cells and organisms in terms of the properties of individual moleculesand ions. For example, the properties of one such molecule,water, have controlled and continue to control the evolution, structure, and function of cells. You cannot understand biology without appreciating how the properties of water control the chemistry of life. Life first arose in a watery environment. Constituting 70-80 percent by weight of most cells, water is the most abundant molecule in biological systems. It is within this aqueous milieu that small molecules and ions, which make up about 7 percent of the weight of living matter, assembleinto the larger macromoleculesand macromolecular aggregatesthat make up a cell's machinery and architecture and so the remaining mass of organisms.

These small molecules include amino acids (the building blocks of proteins), nucleotides (the building blocks of DNA and RNA), lipids (the building blocks of biomembranes), and sugars (the building blocks of starchesand cellulose). Many biomolecules(e.g.,sugars)readily dissolvein water; these molecules are called hydrophilic (water liking). Others (e.g., cholesterol)are oilS fatlike substancesthat shun water; these are said to be hydrophobic (water fearing). Still other biomolecules(e.g.,phospholipids)are a bit schizophrenic, containing both hydrophilic and hydrophobic regions; these molecule are said to be amphipathic. Phospholipids are used to build the flexible membranes that form the wall-like boundaries of cells and their internal organelles.The smooth functioning of cells, tissues' and organisms depends on all these molecules, from the smallest to the largest. Indeed, the chemistry of the simple proton (H*) can be as important to the survival of a human cell as that of each gigantic, genetic-code-carrying DNA molecule (the mass of the DNA molecule in human

OUTLINE 2.1

CovalentBondsand NoncovalentInteractions 32

2.2

of Cells BuildingBlocks Chemical

40

2.3

Equilibrium Chemical

49

2.4

Energetics Biochemical

54

31

(a) Molecularcomplementarity

(bl Chemicalbuilding blocks

ProteinA

Polymerization p&. d'

",llrill t

1

ProteinB

S m a l lm o l e c u l e subunits (dl Chemicalbond energy

{c} Chemicalequilibrium

e#

Macromolecule

"High-energy" phosphoa n h y dr i d e bonds

kr Kl

K^^_

K1

Adenosine triphosphate

k,

A FIGURE2-1 Chemistry of life: four key concepts.(a) Molecular complementarity liesat the heartof all biomolecular interactions, as when two proteinswith complementary shapesand chemical propertiescometogetherto form a tightlybound complex (b) Small moleculesserveas buildingblocksfor IargerstructuresForexample, to generatethe information-carrying macromolecule DNA,four small nucleotidebuildingblocksare covalentlylinkedinto long strings (polymers), which then wrap aroundeachother to form the double helix (c) Chemicalreactionsare reversible, and the distributionof the chemicalsbetweenstartingreagents(/eft)and the productsof the

(nght)depends reactions on the rateconstants of the forward(k1, (k,,lowerarrow)reactions upperarrow)andreverse Theratioof these,K"o,provides an informative measure of the relative amounts (d)In of products andreactants thatwill be present at equilibrium manycases, the source of energyfor chemical reactions in cellsisthe hydrolysis of the molecule ATPThisenergyisreleased whena highenergyphosphoanhydride bondlinkingthe B andy phosphates in (red)isbrokenby the addition theATPmolecule of a watermolecule,

c h r o m o s o m e1 i s 8 . 6 x 1 0 r 0 t i m e s t h a t o f a p r o t o n ! ) . T h e chemical interactions of all of these molecules,large and small, with water and with one anothet define the nature of life. Luckily, although many types of biomolecules interacr and react in numerous and complex pathways to form functional cells and organisms, a relatively small number of chemical principles are necessaryto understand cellular processes at the molecularlevel (Figure 2-l).ln this chapter we review these key principles, some of which you already know well. Sfe begin with the covalent bonds that connect atoms into a molecule and the noncovalent forcesthat stabilize groups of atoms into functional structures within and between molecules. We then consider the key properties of the basic chemical building blocks of macromoleculesand macromolecular assemblies.After reviewing those aspectsof chemical equilibrium that are most relevant to biological systems,we end the chapter with basic principles of bio-

chemical energetics,including the central role of ATP (adenosinetriphosphate) in capturing and transferring energy in cellular metabolism.

32

cHAPTER2 |

CHEMICALFOUNDATTONS

TOrmlnOAUI'311O l',.

A

CovalentBondsand

NoncovalentInteractions Strong and weak attractive forces between atoms are the "glue" that holds them together in individual moleculesand permits interactions between different biomolecules.Strong forces form a covalent bond when two atoms share one pair of electrons("single" bond) or multiple pairs of electrons ("double" bond, "triple" bond, etc.). The weak attractive forces of noncovalent interactions are equally important in determining the properties and functions of biomolecules such as proteins, nucleic acids, carbohydrates,and lipids. .We will first review covalent bonds and then discussthe four

Electrons

Covalentbond H

H

H

H

A FIGURE 2-2 Covalentbondsform by the sharingof electrons. Covalent bonds,the strongforcesthat holdatomstogetherinto molecules, formwhenatomsshareelectrons fromtherroutermost electron orbitals. Eachatomformsa definednumberandqeometrv of covalent bonds

major types of noncovalent interactions:ionic bonds, hydrogen bonds, van der Waals interactions,and the hydrophobic effect.

The ElectronicStructureof an Atom D e t e r m i n e st h e N u m b e ra n d G e o m e t r y of CovalentBondslt Can Make Hydrogen, oxygen, carbon, nitrogen,phosphorus,and sulfur are the most abundant elementsin biological molecules. Theseatoms, which rarely exist as isolatedentities,readily form covalent bonds, using electronsin the outermost electron orbitals surrounding their nuclei (Figure 2-2\. As a rule, each type of atom forms a characteristicnumber of covalent bonds with other atoms, with a well-defined geometry determined by the atom's size and by both the distribution of electrons around the nucleus and the number of electronsthat it can share.In some cases(e.g.,carbon), the number of stable covalent bonds formed is fixed; in other cases (e.g., sulfur), different numbers of stable covalent bonds are possible. All the biological building blocks are organizedaround the carbon atom, which normally forms four covalent bonds with three or four other atoms. As illustrated in Figure 2-3a for formaldehyde, carbon can bond to three atoms, all in a common plane. The carbon atom forms two typical single bonds with two atoms and a double bond (two shared electron pairs) with the third atom. In the absenceof other constraints, atoms joined by a single bond generally can rotate freely about the bond axis, whereas those connected by a double bond cannot. The rigid planarity imposed by double bonds has enormous significancefor the shapesand flexibility of biomoleculessuch as phospholipids, proteins, and nucleic acids. Carbon can also bond to four rather than three atoms. As illustrated by the methane (CHa) molecule,when carbon is bonded to four other atoms, the angle between any two bonds is 109.5' and the positions of bonded atoms define the four points of a tetrahedron(Figure2-3b). This geome-

try defines the structures of many biomolecules.A carbon (or any other) atom bonded to four dissimilar atoms or groups in a nonplanar configuration is said to be asymmetric. The tetrahedral orientation of bonds formed by an asymmetric carbon atom can be arranged in three-dimensional space in two different ways, producing molecules that are mirror imagesof eachother, a property calledchirality (from the Greek word cheir,meaning "hand") (Figure2-4). Such moleculesare called optical isomers,or stereoisomers.Many molecules in cells contain at least one asymmetric carbon atom, often called a chiral carbon atom. The different stereoisomersof a molecule usually have completely different biological activities becausethe arrangement of atoms within their structures differs, yielding their unique abilities to interact and chemically react with other molecules. Some drugs are mixtures of the stereoisomersof small moleculesin which only one stereoisomerhas the biological activity of interest.The use of a pure single stereoisomer of the chemical in place of the mixture can result in a more potent drug with reduced sideeffects.For example,one stereoisomerof the antidepressantdrug citalopram (Celexa) is

(al Formaldehyde

H

c:o H

(b) Methane H

I

H-C-H

I

H Chemical structure

Ball-and-stick model

Space-filling model

2-3 Geometryof bondswhen carbonis covalently A FIGURE linked to three or four other atoms.(a)A carbonatomcanbe (CHzO). Inthiscase,the bondedto threeatoms,asin formaldehyde participate in two singlebondsandone electrons carbon-bonding etoms , h i c ha l ll i ei n t h es a m ep l a n eU. n l i k a d o u b l eb o n dw canrotatef reelyaboutthe by a singlebond,whichusually connected by a doublebondcannot(b)Whena bondaxis,thoseconnected (CH+), the carbonatomformsfoursinglebonds,asin methane (all in the formof in space oriented case) are H in this atoms bonded indicates on the leftclearly Theletterrepresentation a tetrahedron. andthe bondingpattern of the molecule theatomiccomposition the geometric modelin the centerillustrates Theball-and-stick of the balls the diameters bonds, but and of the atoms arrangement are electrons the atomsandtheirnonbonding representing with the bondlengthsThesizesof the smallcompared unrealistically modelon the rightmore cloudsin the space-filling electron in threedimensions thestructure represent accuratelv C O V A L E N TB O N D SA N D N O N C O V A L E N ITN T E R A C T I O N S

33

FIGURE 2-4 Stereoisomers. Manymolecules in cellscontain at leastoneasymmetric carbonatom Thetetrahedral orientation of bondsformedbyan asymmetric carbonatomcanbearranged in three-dimensional spacein two different ways,producing molecules that aremirrorimages, or stereoisomers, of eachother.Shownhereis thecommonstructure of an aminoacid,with itscentral asymmetric carbonandfourattached groups, including the Rgroup,discussed in Section 2 2. Aminoacidscanexistin two mirror-image forms, designated r ando. Although properties thechemical of such stereoisomers areidentical, theirbiological activities aredistinctOnly r aminoacidsarefoundin oroterns

that can participate in noncovalent interactions. Sulfur forms two covalent bonds in hydrogen sulfide (H2S)but also can accommodate six covalent bonds, as in sulfuric acid (H2SO4)and its sulfate derivatives.Nitrogen and phosphorus each have five electronsto share.In ammonia (NH3), the nitrogen atom forms three covalent bonds; the pair of electrons around the atom not involved in a covalent bond can take part in noncovalent interactions. In the ammonium ion (NH+*), nitrogen forms four covalent bonds, which have a tetrahedral geometry. Phosphoruscommonly forms five covalent bonds, as in phosphoric acid (H3POa) and its phosphate derivatives,which form the backbone of nucleic acids. Phosphategroups covalently attachedto proteins play a key role in regulating the activity of many proteins, and the central moleculein cellular energetics,ATP, contains three phosphate groups (seeSection 2.4). A summary of common covalent linkagesand functional groups (portions of molecules that confer distinctive chemical properties) is provided in Table 2-2.

ElectronsMay Be SharedEqually o r U n e q u a l l yi n C o v a l e n tB o n d s

The extent of an atom's ability to attract an electron is called its electronegatiuity. ln a bond between atoms with identical or similar electronegativities,the bonding electrons are es170 times more potent than the other. Somestereoisomershave sentially sharedequally betweenthe two atoms, as is the case very different activities. Darvon is a pain reliever,whereas its for most C-C and C-H bonds. Such bonds are called nonstereoisomer,Novrad (Daruon spelled backward), is a cough polar. In many molecules,the bonded atoms have different suppressant. One stereoisomer of ketamine is an anesthetic, electronegativities,resulting in unequal sharing of the elecwhereas the other causeshallucinations. I trons. The bond betweenthem is said to be polar. One end of a polar bond has a partial negativecharge The number of covalent bonds formed by other common (6-), and the other end has a partial positive charge ( 6+). atoms is shown in Table 2-1,.A hydrogen atom forms only In an O-H bond, for example, the greater electronegativone covalent bond. An atom of oxygen usually forms only ity of the oxygen atom relative to hydrogen results in the two covalent bonds but has two additional pairs of electrons electrons spending more time around the oxygen atom than the hydrogen. Thus the O-H bond possessesan electric dipole, a positive charge separated from an equal but opposite negative charge. The amount of 6- charge on the oxygen atom of a O-H dipole is approximately 25 percent of that of an electron, with an equivalent 6+ charge on the H atom. Becauseof its two O-H bonds that are nor AT()M AIIO USUAI. I,IUMBTB TYPICAL I)UTER TI.ICIROI'IS {]FCl)VATElrlT BI]NDS Bt]I'IIl GIOMETBY on exact opposite sides of the O atom, water molecules (HzO) are dipoles (Figure 2-5) that form electrostatic,noncovalent interactions with one another and with other molH I H ecules. These interactions play a critical role in almost o z every biochemical interaction and so are fundamental to ,ror cell biology. S 214,or5 St The polarity of the O:P double bond in H3PO4 results ,' in a resonancehybrid, a structure between the two forms N shown below in which nonbonding electrons are shown as 3or4 pairs of dots:

-T-

5

P

4

-'rP

I

-?-

H

CHAPTER2 I

CHEMICALFOUNDATIONS

I

:o

o I

H-O-P-O-H il"

o.

34

H

I

e

l*

H-O-P-O-H

I

o

FUNCTIONAL GROUPS

o -c-

o -c-o-

Carbonyl

Carboxyl

(ketone)

( c a r b o x y l i ca c i d )

o -o-P-oI

oPhosphate

oo

iltl -o-P-o-P-

o-

o-

Pyrophosphate (phosphorylated molecule)

(diphosphate)

LINKAGES

o til -c-o-cI

o -N-C-

ll

I Amide

In the resonancehybrid on the right, one of the electrons from the P:O double bond has accumulatedaround the O atom, giving it a negativecharge and leaving the P atom with a positive charge. These chargesare important in noncovalent interactions.

C o v a l e n tB o n d sA r e M u c h S t r o n g e r and More StableThan NoncovalentInteractions Covalentbonds are very stable(i.e.,consideredto be strong) becausethe energies required to break them are much greater than the thermal energy available at room temperature (25 "C) or body temperature(37'C). For example,the

ol

H

2-5 The dipolenatureof a water molecule.The A FIGURE a partialcharge(aweakerchargethantheone symbolE represents proton) in the a Because of thedifference on an electron or of H andO, eachof the polarH-O bondsin electronegativities of eachof anddirections of the dipoles waterisa dipoleThesizes the netdrstance andamountof charqe the bondsdetermine separation, or dipolemoment,of the molecule

thermal energy at25 "C is approximately 0.6 kilocalorie per mole (kcal/mol), whereas the energy required to break the carbon-carbonsingle bond (C-C) in ethane is about 140 times larger (Figure 2-6). Consequently' at room temperature (25 "C), fewer than f. in 1012ethanemoleculesis broken into a pair of 'CH3 molecules,each containing an unpaired, nonbondingelectron(calleda radical). Covalent single bonds in biological moleculeshave energies similar to the energy of the C-C bond in ethane. Because more electrons are shared between atoms in double bonds, they require more energy to break than single bonds. For instance, it takes 84 kcal/mol to break a single C-O bond but 170 kcal/mol to break a C:O double bond' The most common double bonds in biological molecules are C:O, C:N, C:C, and P:O. In contrast, the energy required to break noncovalent interactions is only 1-5 kcalimol, much less than the bond energiesof covalent bonds (seeFigure 2-5). Indeed' noncovalent interactions are weak enough that they are constantly being formed and broken at room temperature' Although these interactions are weak and have a transient existenceat physiologicaltemperatures(25-37 "C)' multiple noncovalent interactions can, as we will see' act together to produce highly stable and specific associations between different parts of alatge molecule or between different macromolecules. Below, we review the four main types of noncovalent interactions and then consider their roles in the binding of biomoleculesto one another and to other molecules. C O V A L E N TB O N D SA N D N O N C O V A L E N ITN T E R A C T I O N S

35

> FIGURE 2-6 Relativeenergiesof covalent Noncovalent interactions bondsand noncovalentinteractions. Bond lonic energies aredefinedastheenergyrequired to breaka particular typeof linkageCovalent Hydrogen b o n d si ,n c l u d i nt g bonds h o s ef o r s i n g l (eC - C ) a n d double(C:C) carbon-carbon bonds.areoneto Thermal two powersof 10stronger thannoncovalent energy interactions Thelatteraresomewhat greater thanthethermalenergyof theenvironment at (25"C) Many normalroomtemperature processes biological arecoupled to the energy 0.24 released duringhydrolysis of a phosphoanhydride bondin ATP

Covalentbonds

Hydrolysisof ATP p h o s p h o a n h y d r i dbeo n d

c-c

C=C

240 kcal/mol

Increasingbond strength

lonic InteractionsAre Attractions between OppositelyChargedlons Ionic interactions result from the attraction of a positively charged ion-a cation-for a negatively charged ion-an anion. In sodium chloride (NaCl), for example, the bonding electron contributed by the sodium atom is completely transferred to the chlorine arom. (Figure 2-7a). Unlike covalent bonds, ionic interactionsdo not have fixed or specificgeometric orientations becausethe electrostatic field around an ion-its attraction for an opposite charge-is uniform in all directions. In solid NaCl, many ions pack tightly together in an alternating pattern to permit opposite charges to align and thus form a highly orderedcrystallinearray (saltcrystals)(Figure2-7b). tVhen solid saltsdissolvein water, the ions separatefrom one another and are stabilized by their interactionswith water molecules.In aqueoussolutions,simpleions of biological

(al

significance,suchas Na*, K*, Ca2*,Mg2* rand Cl-, are hydrated, surrounded by a stable shell of water moleculesheld in place by ionic interactions betweenthe central ion and the oppositely charged end of the water dipole (Figure 2-7c). Most ionic compounds dissolvereadily in water becausethe energy of hydration, the energy releasedwhen ions tightly bind water molecules,is greater than the lattice energy that stabilizesthe crystal structure. Parts or all of the aqueousbydration shell must be removed from ions when they directly interact with proteins. For example, water of hydration is lost when ions pass through protein pores in the cell membrane during nerve conduction. The relative strength of the interaction betweentwo ions, A- and C-, dependson the concentration of other ions in a solution. The higher the concentration of other ions (e.g., Na* and Cl-), the more opportunities A and C* have to

(b)

1,' -> ---.+

cl Cl

Donationof electron

.-#

s

-d q{-

+ Hro dissolving +-Crystallizing

-d

U A FIGURE 2-7 Electrostaticinteractionsof oppositelycharged dissolved in water,the ionsseparate andtheircharges, no longer ionsof salt(NaCl)in crystalsand in aqueoussolution.(a)In balanced by immediately adjacent ionsof opposite charge, are crystalline tablesalt,sodiumatomsarepositively charged stabilized ions(Na+) by interactions with polarwaterWatermolecules andthe dueto the lossof oneelectron each,whereas chloride ionsareheldtogetherby electrostatic atomsare interactions between the correspondingly (Ct I 5Ugainingoneelectron negatively charged charges on the ionandthe partialcharges on thewater'soxygenand each(b)Insolidform,ioniccompounds formneatlyordered hydrogen atomsIn aqueous arrays, solutions, all ionsaresurrounded by a or crystals, of tightlypackedionsin whichthe positive andnegatively hydration shellof watermolecules charged ionscounterbalance eachother.(c)Whenthe crvstals are

36

CHAPTER2 I

CHEMICALFOUNDATIONS

(c)

(b)

(a)

:O-H

I

H H

iuH tll O-H

I o-H

H

I :O-H

:O-H

:O-H

H

ll

H-O:

I :O-H

HH H-O

Water-water

: O-H

H-O: I

I

H HH

:o -i-rtr-

:O- CHs

Alcohol-water

I

:N-CHs H

Amine-water

H

H

o -i - o-

.H-O: I H

Peptide group-water

Ester group-water

2-8 Hydrogenbondingof water with itself and with A FIGURE in an outerelectrons Eachpairof nonbonding other compounds. atomin a hydrogen atomcanaccepta hydrogen oxygen or a nitrogen andtheaminogroupscanalsoformhydrogen bond Thehydroxyl forms bondswithwater.(a)In liquidwater,eachwatermolecule others, creating a dynamic hydrogen bondswithseveral transient

(b)Wateralsocanform molecules networkof hydrogen-bonded for the high accounting andamines, bondswith alcohols hydrogen (c)Thepeptide groupandestergroup, of thesecompounds solubility participate in commonly in manybiomolecules, whicharepresent bondswithwateror polargroupsin othermolecules. hydrogen

interact ionically with these other ions and thus the lower the energyrequired to break the interaction betweenA- and C*. As a result, increasingthe concentrationsof saltssuch as NaCl in a solution of biological moleculescan weaken and even disrupt the ionic interactions holding the biomolecules together.

O-H bonds within a singlewater molecule (Figure2-8a). The strengthof a hydrogen bond betweenwater molecules (approximately 5 kcal/mol) is much weaker than a covalent O-H bond (roughly 110 kcal/mol), although it is greater than that for many other hydrogen bonds in biological molecules(t-2 kcall mol). The extensivehydrogen bonding between water molecules accounts for many of the key properties of this compound' including its unusually high melting and boiling points and its ability to interact with (e.g.,dissolve)many other molecules. The solubility of unchargedsubstancesin an aqueousenvironment dependslargely on their ability to form hydrogen bondswith water.For instance,the hydroxyl group (-OH) in an alcohol (XCH2OH) and the amino group (-NH2) in amines (XCH2NH2) can form severalhydrogen bonds with water, enabling these moleculesto dissolvein water to high concentrations(Figure2-8b). In general,moleculeswith polar bonds that easilyform hydrogen bonds with water' as well as charged moleculesand ions that interact with the dipole in water, can readily dissolve in water; that is' they are hydrophilic (water liking). Many biological moleculescontain, in addition to hydroxyl and amino groups' peptide and ester groups, which form hydrogen bonds with water via otherwisenonbondedelectronson their carbonyl oxygens(Figure 2-8c). X-ray crystallography combined with computational analysispermits an accuratedepiction of the distribution of the outermostunbondedelectronsof atoms as well as the electrons in covalent bonds, as illustrated in Figure 2-9.

HydrogenBondsDeterminethe Water S o l u b i l i t yo f U n c h a r g e dM o l e c u l e s A hydrogen bond is the interaction of a partially positively charged hydrogen atom in a molecular dipole (e.9., water) with unpaired electronsfrom another atom, either in the same (intramolecular\or different Untermolecular)molecule.Normall5 a hydrogen atom forms a covalent bond with only one other atom. However, a hydrogen atom covalentlybonded to an electronegativedonor atom D may form an additional weak association,the hydrogen bond, with an acceptoratom A, which must have a nonbonding pair of electronsavailable for the interaction: D6--H6+

+ : 46- i-

D6--H6*:.....:: 46-

uyarolln uona The length of the covalent D-H bond is a bit longer than it would be if there were no hydrogen bond becausethe acceptor "pulls" the hydrogen away from the donor. An important feature of all hydrogen bonds is directionality. In the strongest hydrogen bonds, the donor atom, the hydrogen atom, and the acceptor atom all lie in a straight line. Nonlinear hydrogen bonds are weaker than linear ones; still, multiple nonlinear hydrogen bonds help to stabilize the three-dimensionalstructuresof many proteins. Hydrogen bonds are both longer and weaker than covalent bonds betweenthe same atoms. In water, for example, the distance between the nuclei of the hydrogen and oxygen atoms of adjacent, hydrogen-bondedmoleculesis about 0.27 nm, about twice the length of the covalent

Van der WaalsInteractions Are Causedby TransientDiPoles When any two atoms approach each other closel5 they create a weak, nonspecific attractive force called a van der Waals interaction. Thesenonspecificinteractionsresult from the momentary random fluctuations in the distribution of the electrons of any atom' which give rise to a transtent C O V A L E N TB O N D SA N D N O N C O V A L E N ITN T E R A C T I O N S

37

clouds, the atoms are said to be in van der $7aalscontact. The strength of the van der rWaalsinteraction is about 1 kcal/mol. weaker than typical hydrogen bonds and only slightly higher than the averagethermal energy of moleculesat 25 "C. Thus multiple van der Waals interactions,a van der'Waalsinteraction in conjunction with other noncovalent interactions, or both are required to significantly influencethe stability of inter- and intramolecular conracrs.

The HydrophobicEffectCausesNonpolar Moleculesto Adhere to One Another Becausenonpolar molecules do not contain charged groups, possessa dipole moment, or becomehydrated,they are insoluble or almost insolublein water; that is, they are hydrophobic (water fearing).The covalentbonds betweentwo carbon atoms and between carbon and hydrogen atoms are the most common nonpolar bonds in biological systems.Hydrocarbons-molecules made up only of carbon and hydrogen-are virtually insoluble in water. Large triacylglycerols(or triglycerides),which make up A FIGURE 2-9 Distributionof bondingand outer nonbonding animal fats and vegetableoils, also are insolublein water. As we electronsin the peptidegroup.Shownhereisa peptidebond will see later, the major portion of these moleculesconsistsof linkingtwo aminoacidswithina proteincalledcrambinTheblack long hydrocarbon chains. After being shaken in water, triacyllinesrepresent thecovalent bondsbetween atomsThered(negative) glycerols form a separatephase.A familiar example is the sepaandblue(positive) linesrepresent contours of chargedetermined ration of oil from the water-basedvinegar in an oil-and-vinegar usingx-raycrystallography andcomputational methods. Thegreater salad dressing. the numberof contourlines,the higherthe charge. Thehighdensity Nonpolar moleculesor nonpolar portions of molecules of redcontourlinesbetweenatomsrepresents the covalent bonds (shared tend to aggregatein water owing to a phenomenoncalled pairs). electron Thetwo setsof redcontourlinesemanating fromtheoxygen(O)andnotfallingon a covalent the hydrophobic effect. Becausewater molecules cannot (black bond line) represent thetwo pairsof nonbonded form hydrogen bonds with nonpolar substances,they tend electrons on the oxygen that areavailable to participate in hydrogen bondingThehighdensity to form "cages" of relatiuely rigid hydrogen-bonded of bluecontourlinesnearthe hydrogen (H)bondedto nitrogen (N) pentagons and hexagons around nonpolar molecules represents a partialpositive charge, indicating thatthisH canactasa donorin hydrogen bonding. C Jelsch etal, 2000,procNat,t. [From Acad SciUSA97.3171 CourtesyofM M Teeterl

unequal distribution of electrons. If two noncovalently bonded atoms are close enough together, electrons of one atom will perturb the electronsof the other. This perturbation generatesa transient dipole in the secondatom, and the two dipoles will attract each other weakly (Figure 2-10). Similarly, a polar covalent bond in one molecule will attract an oppositely oriented dipole in another. Van der'Waals interactions, involving either transiently induced or permanent electric dipoles, occur in all types of molecules, both polar and nonpolar. In particular, van der \Waalsinteractions are responsible for the cohesion between nonpolar moleculessuch as heptane,CH3-(CH2)s-CH:, that cannot form hydrogen bonds or ionic interactionswith 'Waals other molecules.The strength of van der interactions decreasesrapidly with increasingdistance;thus thesenoncovalent bonds can form only when atoms are quite closeto one another. However, if atoms get too close together,they become repelled by the negative charges of their electrons. \fhen the van der Waals attraction between rwo aroms exactly balances the repulsion between their two electron

38

CHAPTER2 |

cHEM|CALFOUNDAT|ONS

i<---->i

k----t

Covalent radius ( 0 . 0 6n 2m )

van derWaals radius ( 0 . 1 4n m )

A FIGURE 2-10 Two oxygenmoleculesin van der Waals contact.ln thismodel,redindicates negative chargeandblue positive indicates chargeTransient dipoles in theelectron clouds of all atomsgiveriseto weakattractive forces,calledvander Waals interactions. Eachtypeof atomhasa characteristic vanderWaals radius at whichvanderWaalsinteractions withotheratomsare optimal. Because atomsrepeloneanotherif theyarecloseenough together for theirouterelectrons to overlap withoutbeingshared in a covalent bond,thevanderWaalsradiusisa measure of thesizeof theelectron cloudsurrounding an atom Thecovalent radrus indicated hereisfor thedoublebondof O:O; thesinqle-bond covalent radius of oxygenisslightly longer.

Nonpolar substance

Watersreleasedinto bulk solution H i g h l yo r d e r e d w a t e rm o l e c u l e s

\\ o o'o \.t

o

o Hydrophobic aggregation

@o Lowerentropy

Higherentropy

2-11 Schematic depictionof the hydrophobiceffect' FIGURE in molecules thatformaroundnonpolar Cagesof watermolecules in thesurrounding thanwatermolecules aremoreordered solution r esd u c et h s en u m b e r b u l kl i q u i dA g g r e g a t i o nf n o n p o l amr o l e c u l e resulting in a in highlyordered cages, involved of watermolecules compared state(nErht) moreenergetical lyf avorable higher-entropy, state(/eft) with the unaggregated

(Figure 2-1I, Ieft). This state is energeticallyunfavorable becauseit decreasesthe randomness(entropy) of the population of water molecules.(The role of entropy in chemic a l s y s t e m si s d i s c u s s e di n a l a t e r s e c t i o n . )I f n o n p o l a r moleculesin an aqueousenvironment aggregatewith their hydrophobic surfacesfacing each other, the hydrophobic s u r f a c e a r e a e x p o s e dt o w a t e r i s r e d u c e d ( F i g u r e 2 - 1 1 , right). As a consequence,lesswater is neededto form the cages surrounding the nonpolar molecules, and entropy increases(an energeticallymore favorable state)relative to the unaggregatedstate. In a sense,then, water squeezes the nonpolar moleculesinto spontaneouslyforming aggregates.Rather than constituting an attractive force such as in hydrogen bonds, the hydrophobic effect resultsfrom an a v o i d a n c e o f a n u n s t a b l e s t a t e ( e x t e n s i v ew a t e r c a g e s a r o u n d i n d i v i d u a ln o n p o l a r m o l e c u l e s ) . Nonpolar moleculescan also associate,albeit weakly' through van der Vaals interactions.The net result of the hydrophobic and van der Waals interactionsis a very powerful tendencyfor hydrophobic moleculesto interact with one another,not with water. Simply put, like dissolueslike' Polar moleculesdissolve in polar solvents such as water; nonpolar moleculesdissolvein nonpolar solventssuch as hexane.

y ediated M o l e c u l a rC o m p l e m e n t a r i t M PermitsTight, Interactions Noncovalent via H i g h l yS p e c i f i cB i n d i n go f B i o m o l e c u l e s Both inside and outside cells, ions and molecuiesare constantly bumping into one another.The greaterthe number of copiesof any two types of moleculesper unit volume (i'e.,

the higher their concentration)' the more likely they are to encounter one another.Vhen two moleculesencounter each other, they most likely will simply bounce apart becausethe noncovalent interactions that would bind them together are weak and have a transient existenceat physiological temperatures. However, moleculesthat exhibit molecular complementarity, a lock-and-key kind of fit between their shapes, form multiple noncharges,or other physical properties,can '$7hen two such struccovalent interactions at close range. turally complementarymoleculesbump into each other' they can bind (stick) together. Figure 2-12 tllustrates how multiple, different weak bonds can bind two proteins together. Almost any other arrangement of the same groups on the two surfaceswould not allow the molecules to bind so tightly. Such multiple, specificinteractions betweencomplementary regions within a orotein molecule allow it to fold into a unique threedimensionalshape(Chapter 3) and hold the two chains of DNA togetherin a double helix (Chapter 4)' Similar interactionsunderliethe associationof groups of more than two molecules into multimolecular complexes, leading to formation of muscle fibers' to the gluelike associationsbetween cells in solid tissues,and to numerous other cellular structures. Depending on the number and strength of the noncovalent interactions betweenthe two moleculesand on their environment, their binding may be tight (strong) or loose (weak) and, as a consequence,either long lasting or transient.The higher the affinity of two moleculesfor each other, the better the molecular "fit" between them, the more noncovalent interactionscan form, and the tighter they can bind

-cH3 | -CH3 H3( ,

_cH3 H3C_

ProteinB ProteinA Stable complex

ProteinC ProteinA Less stable comPlex

andthe bindingof complementarity 2-12 Molecular FIGURE Thecomplementary proteinsvia multiplenoncovalentinteractions. of two proteinsurfaces polarity, andhydrophobicity charges, shapes, produce a whichin combination weakinteractions, permrt multiple from molecular deviations Because binding. tight and stronginteraction surface a parlicular binding, weaken substantially complementarity canbindtightlyto onlyoneor a usually of anygivenbiomolecule region of the Thecomplementarity of othermolecules verylimitednumber much more bind permits to them left on the two proteinmolecules proteins on therighi tightlythanthetwo noncomplementary INTERACTIONS T C O V A L E N TB O N D SA N D N O N C O V A L E N T

39

together.An important quantitative measureof affinity is the binding dissociationconstant K6, describedlater. As we discussin Chapter 3, nearly all the chemicalreactionsthat occur in cellsalso dependon the binding propertiesof enzymes.Theseproteins not only speedup, or cataIyze, reactions but also do so with a high degree of specificity, a reflection of their ability to bind tightly to only one or a few related molecules. The specificity of intermolecularinteractionsand reactions,which dependson molecular complementarity,is essentialfor many processes critical to life.

Covalent Bonds and Noncovalent Interactions Covalent bonds, which bind the atoms composing a olecule in a fixed orientation, consist of pairs of electrons shared by two atoms. They are stable in biological systems because the relatively high energies required to break them (50-200 kcal/mol) are much larger than the thermal kinetic energyavailableat room (25 .C) or body (37 "C) temperatures. r Many moleculesin cells contain at least one asymmetric carbon atom, which is bonded to four dissimilar atoms. Such moleculescan exist as optical isomers (mirror images),designatedn and r (seeFigure 2-4), which have different biological activities.In biological systems, nearly all sugarsare D isomers,whereasnearly all amino acids are L isomers. trons may be sharedequally or unequally in covalent Atoms that differ in electronegativityform polar cobonds in which the bonding electronsare distributed unequally. One end of a polar bond has a partial positive charge and the other end has a partial negativechaige (see Figure2-5). r Noncovalent interactions between atoms are considerably weaker than covalent bonds, with bond energlesranging from about 1-5 kcal/mol (seeFigure 2-6). r Four main types of noncovalent interactions occur in biological systems:ionic bonds, hydrogen bonds, van der \faals interactions, and interactions due to the hvdroohobic effect. r Ionic bonds result from the electrostatic attraction between the positive and negativechargesof ions. In aqueous solutions, all cations and anions are surrounded by a shell of bound water molecules(seeFigure Z-7c).Increasingthe salt (e.g.,NaCl) concentrarionof a solutioncan weakenthe relative strength of and even break the ionic bonds between biomolecules. hydrogen bond, a hydrogen atom covalently bonded electronegativeatom associateswith an accepror whose nonbonding electrons attract the hydrogen gure 2-8).

40

CHAPTER2 |

cHEMTCALFOUNDATTONS

r Weak and relatively nonspecificvan der'Sfaalsinteractions are createdwheneverany two atoms approach each other closely. They result from the attraction between transient dipoles associatedwith all molecules (seeFigure2-10). r In an aqueousenvironment, nonpolar moleculesor nonpolar portions of larger molecules are driven together by the hydrophobic effect, thereby reducing the exrent of their direct contactwith water molecules(seeFigure2-11). r Molecular complementarity is the lock-and-key fit between moleculeswhose shapes,charges,and other physical properties are complementary.Multiple noncovalent interactions can form betweencomplementary molecules,causing them to bind tightly (seeFigure2-12),but not between moleculesthat are not complementary. r The high degreeof binding specificity that results from molecular complementarity is one of the features that underlies intermolecular interactions and thus is essentialfor many processescritical to life.

E

Chemical BuildingBlocksof Cells

A common theme in biology is the construction of large molecules (macromolecules)and structures by the covalent or noncovalent associationof many similar or identical smaller molecules.The three most abundant classesof the critically important biological macromolecules-proteins, nucleic acids, and polysaccharides-are all polymers composed of multiple covalently linked building block small molecules,or monomers (Figure 2-13). Proteins are linear polymers containing 10 to severalthousand amino acids linked by peptide bonds. Nucleic acids are linear polymers containing hundreds to millions of nucleotides linked by phosphodiester bonds. Polysaccharidesare linear or branched polymers of monosaccharides(sugars)such as glucose linked by glycosidic bonds. Although the actual mechanismsby which covalent bonds betweenmonomers form are complex and will be discussedlater, the formation of a covalent bond between two monomer molecules usually involves the net loss of a hydrogen (H) from one monomer and a hydroxyl (OH) from the other monomer-or the net loss of one water-and can therefore be thought of as a debydration reaction. These bonds are stable under normal biological conditions (e.g., 37"C, neutral pH), and so thesebiopolymers are stable and can perform a wide variety of jobs in cells (store information, catalyze chemical reactions, serve as structural elements in defining cell shapeand movement, etc.). Macromolecular structures can also be assembledusing noncovalent interactions. The macromolecular two-layered (bilayer) structure of cellular membranesis built uo bv the noncovalent assemblyof many thousandsof small molecules called phospholipids (seeFigure 2-13).In this chapter, we will focus on the characteristicsof the monomeric chemical

POLYMERS

MONOMERS

ttl

H2N-C -C-OH I R

HHOHHOHHOHHO

HO

HO +

ttl

|

I R

I

l

ll,l

L ll

N-C-CrN-C-C-OH I I | R* R3 I

peptidebond

Amino acid

o tl

| ll I

| ll |

H-N-C-ciN-C-C | I lt R2 R, l"

oH

H-N-C-C-

PolYPePtide

B L

Base

?1,

HO-P-O

I

HO-P-OJs,

.OH

6Nucleic acid

Nucleotide

glYcosidicbond

Polysaccharide

Monosaccharide

I Hyoropr'irt. h e a dg r o u p J

Phospholipid

2-13 Overviewof the cell'sprincipalchemical FIGURE threemajortypesof biological buifdingblocks.(Top)The of bythe polymerization assembled are each macromolecules (monomers) type:proteins of a particular smallmolecules multiple acidsfromnucleotides 3),nucleic fromaminoacids(Chapter building blocks-amino acids,nucleotides,sugars'and phospholipids. The structure, function, and assemblyof proteins, nucleic acids, polysaccharides,and biomembranes are discussedin subsequentchapters.

A m i n o A c i d sD i f f e r i n gO n l y i n T h e i r SideChainsComposeProteins The monomeric building blocks of proteins ate 20 amino acids, which when incorporated into a protein polymer are sometimescalled residues.All amino acidshave a characteristic structureconsistingof a central alpha (cr)carbon atom (C') bonded to four different chemical groups: an amino (NHz)

(sugars)Each from monosaccharides (Chapter 4), andpolysaccharides whose reaction polymer by a linkedintothe iscovalently monomer (Boftom) In (dehydration) molecule net resultis lossof a water into a bilayer assemble noncovalently monomers phospholipid contrast, (chapter10) membranes of allcellular whichformsthebasis structure, group, a carboxylic acid or carboxyl (COOH) group (hence amino acid),a hydrogen (H) atom, and one variable Ih. *-. group, called a side chain or R group' Becausethe ct carbon i.-nali amino acids except glycine is asymmetric,thesemolecules can exist in two mirror-image forms called by convention the I (dextro) and the r- (levo) isomers (seeFigure 2-4)' The two isomerscannot be interconverted(one made identical with the other) without breaking and then re-forming a chemical bond in one of them. !7ith rare exceptions, only the I- forms of amino acids are found in proteins' To understand the three-dimensional structures and functions of proteins' discussedin detail in Chapter 3, you must be familiar with some of the distinctive properties of

B U I L D I N GB L O C K SO F C E L L S CHEMICAL

41

H Y D R O P H O BA I CM I N OA C I D S

cooI

*H3N-C-H I cHs

coo-

+ H . N - c I- H

"l

CH HsC

cooI

+H3N-c-H

CHs

cooI

cooI

+H3N-c-H

cooI

cooI

'H"N-C-H

-l

cooI

*H.N-C-H

-l

H-C - CH.

CH,

CH,

CH,

CH

C:CH

t"

lcHs

t-

HaC

t-

CHs

Lr*t OH

\/ Alanine ( A l ao r A l

Valine (Val orVl

lsoleucine ( l l eo r l l

Leucine (Leu or Ll

H Y D R O P H I LA I CM I N OA C I D S

Methionine (Met or M)

Acidic amino acids

cooI

coo-

I +H3N-c-H

I CHt

CH,

l-

NHs*

Lysine (Lys or Kl

CH,

t-

CH, lCH,

tCH, t-

Glycine (Gty or G)

.

H-C-OH

coo-

oH

cHs

Aspartate (Asp or Dl

Serine (Ser or Sl

Threonine (Thr orT)

I

'H"N-C-H

-l

CH,

IcH, )' cooGlutamate ( G l uo r E )

proline (pro or p)

amino acids, which are determined in part by their side chains. You need not memorize the detailed structure of each type of side chain to understandhow proteins work because amino acids can be classified into several broad categories basedon the size,shape,charge,hydrophobicity (a measure of water solubility), and chemical reactivity of the side chains (Figure 2-74). However, you should be familiar with the generalproperties of each category. Amino acids with nonpolar side chains are hydrophobic and so poorly soluble in water. The larger the nonpolar side chain, the more hydrophobic-less water soluble-the amino acid. The noncyclic side chains of alanine, ualine, leucine, and. isoleucine(calledaliphatic),as well as methionine,consisren_ tirely of hydrocarbons, except for the one sulfur arom in me_ thionine, and all are nonpolar. phenylalanine, tyrosine, and. 42

r"l t-i

coo+H3N-C-H

coo-

l-

NH i C: NHr+ lNHz Arginine (Argor Rl

SPECIALAMINO ACIDS

Cysteine (Cys or C)

cootl

CH,

CH,

Tryptophan (TrporW)

Polar amino acids with uncharged R groups

*H3N-C-H

I 'H"N-C-H -l

lCH,

Tyrosine (TyrorYl

+H.N-c-H

-l

coo-

Phenylalanine (Phe or Fl

c H A p r E R 2| c H E M t c A L F o u N D A T t o N s

FIGURE 2-14 The 20 commonaminoacidsusedto build proteins.Thesidechain(Rgroup;red)determines thecharacteristic properties of eachaminoacidandisthe basisfor grouping amino acidsintothreemaincategories: hydrophobic, hydrophilic, and special. Shownarethe ionized formsthatexistat the pH(=7)of the cytosolIn parentheses arethethree-letter andone-letter abbreviations for eachaminoacid.

tryptophan have large, bulky aromatic side chains. In later chapters, we will see in detail how these hydrophobic side chains under the influence of the hydrophobic effeci often pack in the interior of proteins or line the surfacesof proteins that are embeddedwithin hydrophobic regions of biomembranes. Amino acids with polar side chains are hydrophilic; the most hydrophilic of theseamino acids is the subsetwith side chains that are charged (ionized) at the pH typical of biological fluids (=7)-both inside and outside the cell (seeSectio-n 2.3). Arginine and lysine have positively charged side chains and are called basic amino acids;aspartic acid and glutamic acid have negatively charged side chains due to the carboxylic acid groups in their side chains (their charged forms are called aspartate and glutamate) and are called acidic. A fifth amino acid, histidine, has a side chain containing a ring

with two nitrogens, called imidazole, which can shift from being positively charged to uncharged depending on small changesin the acidity of its environment:

I

A c e t y ll y s i n e

C H 3 - C - N - C H 2 - C H 2 - C H 2 - C H " - C H - C-Ol O -

NH.-

o - o - Ptl o - c H 2 - c H - c o o -

ll Phosphoserine

ll O

NH"OH

pH 5.8

PH 7.8

The activities of many proteins are modulated by shifts in environmental acidity through protonation or deprotonation of histidine side chains. Asparagine and glutamine are uncharged but have polar side chains containing amide groups with extensivehydrogen-bondingcapacities'Similarl5 serine and threonine are unchargedbut have polar hydroxyl groups, which also participate in hydrogen bonds with other polar molecules. Lastl5 cysteine,glycine, and proline exhibit specialroles in proteins becauseof the unique properties of their side chains. The side chain of cysteine contains a reactive sulfhydryl group (-SH), which can oxidize to form a covato a secondcysteine: lent disulfide bond (-S-S-/

I I- H

I

I H,9-9H

3-Hydroxyproline

II H2C\ * N //CH-COOH, HC:C-CH2-CH-COOtrl NH.H3C-N-C-.N

3-Methylhistidine

H

-ooc y-Carboxyglutamate

-OOC

c H - c H' "l - c H - c o o ilH.*

of aminoacidside 2-15 €ommonmodifications A FIGURE others andnumerous residues chainsin proteins.Thesemodified groups(red)to theamino chemical of various areformedby addition chain of a polypeptide acidsidechainsduringor aftersynthesis

N-H

N-H

I

H-C-CH2-

SH+ HS-CH2q

ll

C:O

C:O

reveals that they contain upward of 100 different amino acids.Chemical modifications of the amino acids account for

ll

tl H-N

r''l ll

H-C-

ll O:C ll

C H 2- S - S -

N-H C H 2- c - H

C:O

Regionswithin a singleprotein chain (intramolecular) or in separatechains (intermolecular) sometimesare crosslinked through disulfide bonds. Disulfide bonds stabilizethe folded structure of some proteins. The smallestamino acid, glycine, has a singlehydrogen atom as its R group. Its small sizeallows it to fit into tight spaces.Unlike the other common amino acids, the side chain of proline bends around to form a ring by covalently bonding to the nitrogen atom (amino group) attached to the Co. As a result' proline is very rigid and createsa fixed kink in a protein chain, limiting how a protein can fold in the region of proline residues. Some amino acids are more abundant in proteins than others. Cysteine,tryptophan, and methionine are rare amino acids: together they constitute approximately 5 percent of the amino acids in a protein. Four amino acids-leucine, serine, lysine, and glutamic acid-are the most abundant amino acids, totaling 32 percent of all the amino acid residuesin a typical protein. However, the amino acid composition of proteins can vary widely from thesevalues. Although cellsusethe 20 amino acidsshown in Figure2-14 inthe initial synthesisof proteins' analysisof cellular proteins

and plants but less studied-perhaps becauseof the relative insta-bilityof phosphorylatedhistidine-and apparently rare in mammals.ltt. iia. chains of asparagine,serine,and threonine are sitesfor glycosylation,the attachmentof linear and branched carbohydrate chains. Many secretedproteins and membrane proteins contain glycosylated residues' Other amino acid modifications found in selectedproteins include the hydroxylation of proline and lysine residuesin collagen (Chapter 19), the methylation of histidine residuesin memb."ne rec.ptors' and the 7 carboxylation of glutamate in blood-clotting factors such as prothrombin' Acetylation, addition of an acetyl group' to the amino gro,rp oi the N-terminal residue, is the most common form acid chemical modification, affecting an estimated 6f "-ino 80 percentof all proteins:

o lllll

RO

cH3-c- r y- g- cll HH Acetylated N-terminus OF CELLS B U I L D I N GB L O C K S CHEMICAL

O

43

This modification may play an important role in controlling the life span of proteins within cells becausenonacetylateJ proteins are rapidly degraded.

PURINES NH,

O

ltl

Five Different Nucleotides A r e U s e dt o B u i l d N u c l e i cA c i d s Two types of chemically similar nucleic acids, DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). are the principal genetic-information-carryingmoleculesof the cell. The monomers from which DNA and RNA polymers are built, called nucleotides,all have a common structure: a phosphategroup linked by a phosphoesterbond to a pentose (a five-carbon sugar molecule) that in turn is linked io a nitrogen- and carbon-containing ring structure commonly referred to as a base (Figure 2-16a).In RNA, the pentoseis ribose; in DNA, it is deoxyribose that at posirion 2, has a proton rather than the hydroxyl group at that site in ribose

HH Adenine (A)

(Gl Guanine

PYRIMIDINES NH,

il

tru/?--cH

r"^tl

.r'"'ii" vl H Cytosine (C)

A FIGURE 2-17 Chemical structuresof the principalbasesin nucleicacids.ln nucleic acidsandnucleotides, nitrogen 9 of purines andnitrogen1 of pyrimidines (red)arebondedto the 1, carbonof riboseor deoxyribose U isonlyin RNA,andT isonlyin DNA Both R N Aa n dD N Ac o n t a iA n , G ,a n dC .

position 1 of a pyrimidine (N1). The acidic character of

which form ionic interactions with the negatively charged phosphates. Cells and extracellular fluids in organismscontain small concentrationsof nucleosides,combinations of a baseand a sugar without a phosphate.Nucleotides are nucleosidesthat have one, two, or three phosphate groups esterified at the 5' hydroxyl. Nucleoside monophosphateshave a single esterified phosphate (seeFigure 2-16a); nucleoside diphosphates contain a pyrophosphategroup:

oo

-o-l-o-A-o-

(a) Adenine

Nhz l

(b)

'-c'

5',

N ; , 6 ; -c

\

HOCH2

daH HC.1 : 1c s,/ -'N' N

o - o - P - O - c H5',

o-

5',

HOCH2

Phosphate 2'

OH

H

Ribose Adenosine 5'-monophosphate (AMP}

2-Deoxyribose

FIGURE 2-15 Commonstructureof nucleotides. (a)Adenosine 5' -monophosphate (AMp),a nucleotide present in RNAByconvention, thecarbonatomsof the pentose sugarin nucleotides arenumbered with primes. In naturalnucleotides, the 1, carbonisjoinedby a B linkage to the base(inthiscaseadenine); boththe base(blue)and the phosphate on the 5, hydroxyl (red)extendabovethe planeof the sugarring.(b)Ribose anddeoxyribose, the pentoses in RNAand DNA,respectively 44

.

c H A p r E R 2| c H E M t c A L F o u N D A T t o N S

o-

o-

Pyrophosphate

and nucleosidetriphosphateshave a third phosphate.Table 2-3 lists the names of the nucleosidesand nucleotidesin nucleic acids and the various forms of nucleosidephosphates.The nucleosidetriphosphatesare used in the synthesisof nucleic acids,which we cover in Chapter 4. Among their other functions in the cell, GTP participares in intracellular signaling a_ndacts as an energy reservoir,particularly in protein synthesis, and ATP, discussedlater in this chapter, is the most widely usedbiologicalenergycarrier.

M o n o s a c c h a r i d eJso i n e db y G l y c o s i d i B c onds Form Linearand Branchedpolysaccharides The building blocks of the polysaccharidesare rhe simple sugars, or monosaccharides.Monosaccharides are carbohy_ drates,which are literally covalently bonded combinationsof carbon and water in a one-to-one ratio (CH2O),, where n e q u a l s3 , 4 , 5 , 6 , o r 7 . H e x o s e s( n : 6 ) a n d p e n t o s e (sn : 5 ) are the most common monosaccharides. All monosaccharides

GUANINT(G}

ADENINE(A)

nNe Jin

t,,'o^o

innNe f Iino*e

uBACrr(lJ) THYMINE(I)

cYT0srNt(q

Adenosine

Guanosine

Cytidine

Uridine

Deoxyadenosine

Deoxyguanosine

Deoxycytidine

DeoxYthYmidine

Adenylate

Guanylate

Cytidylate

UridYlate

Deoxycytidylate

Deoxythymidylate

Deoxyadenylate

DeoxyguanYlate

Nucleoside monophosphates

AMP

GMP

CMP

UMP

Nucieosidediphosphates

ADP

GDP

CDP

UDP

Nucleoside triphosphates

ATP

GTP

CTP

UTP

Deoxynucleoside mono-, di-, and triphosphates

dAMP,etc.

dGMP,etc'

dCMP, etc

dTMP' etc.

contain hydroxyl (-OH) a keto group:

oo

- c t- icl - H lll

Aldehyde

groups and either an aldehyde or

Htt,

6

(-rQ

n-J'-oH

H

- c -Lcf - c - l

1

<_

HO-J.-H

Keto HOH

Many biologically important sugarsare hexoses,including glucose,mannose,and galactose(Figure2-18). Mannose is identical with glucose except that the orientation of the groups bonded to carbon 2 is reversed.SimilarlS galactose, another hexose,differs from glucoseonly in the orientation of the groups attached to carbon 4. Interconversion of glucose and mannose or galactose requires the breaking and making of covalent bonds; such reactions are carried out by enzymescaIIedepimerases. l-Glucose (C6H12O6)is the principal externalsourceof energy for most cells in higher organisms and can exist in three different forms: a linear structure and two different hemiacetalring structures(Figure 2-18a).lf the aldehyde group on carbon 1 reactswith the hydroxyl group on carbon 5, the resulting hemiacetal,o-glucopyranose'contains a sixmember ring. In the cr anomer of o-glucopyranose,the hydroxyl group attachedto carbon 1 points "downward" from the ring as shown in Figure 2-18a; in the B anomer, this hydroxyl points "upward." In aqueoussolution the ct and B anomers readily interconvert spontaneously;at equilibrium there is about one-third ct anomer and two-thirds B, with very little of the open-chain form. Becauseenzymescan distinguish between the ct and B anomers of n-glucose, these forms have distinct bioloeical roles. Condensationof the

lo H-C-OH HOH

H- lLoH

o-Glucofuranose (rarel

o-Glucopyranose (common)

ol -cH20H o-Glucose

(b)

H,r, rO (^.

Ht,ro (-

Ho-l'?-H

H-l'-oH HO-ILH

HO-ILH

H- lLos H-lu-os

ulr,,o,-,

o-Mannose

HO-JLH H-CLoH

ul*ro*

D-Galactose

structuresof hexoses'All hexoses 2-18 Chemical FIGURE andcontainan aldehyde formula(C6H1206) havethe samechemical from aregenerated or a ketogroup.(a)Theringformsof o-glucose the 1 with at carbon aldehyde of the by reaction the linearmolecule readily are forms three 4. The carbon or 5 carbon on hydroxyl in form(right)predominates the pyranose although interconvertible, the (b)In o-mannose ando-galactose, systems. biological andOH(blue)boundto onecarbon of the H (green) configuration exist likeglucose, glucose. Thesesugars, in that atomdiffersfrom primarily asPYranoses. B U I L D I N GB L O C K SO F C E L L S CHEMICAL

.

45

hydroxyl group on carbon 4 of the linear glucose with its aldehydegroup results in the formation of o-glucofuranose. a hemiacetalcontaining a five-memberring. Although ali three forms of n-glucose exist in biological systems,the pyranose form is by far the most abundant. The pyranosering in Figure 2-18a is depictedas planar. In fact, because of the tetrahedral geometry carbon "rorrrd atoms, the most stableconformation of a pyranose ring has a nonplanar, chairlike shape.In this conformation, each bond from a ring carbon to a nonring atom (e.g.,H or O) is either nearly perpendicular to the ring, referred to as axial (a), or nearly in the plane ofthe ring, referredto as equatorial (e):

yranoses tPyranoses

bacteria, and molds produce cellulose-degradingenzymes. Cows and termites can break down cellulose becausethey harbor cellulose-degradingbacteriain their gut. The enzymes that make the glycosidic bonds linking monosaccharidesinto polysaccharidesare specific for the a or B anomer of one sugar and a particular hydroxyl group on the other. In principle, any two sugar molecules can be linked in a variety of ways becauseeachmonosaccharidehas multiple hydroxyl groups that can participate in the formation of glycosidic bonds. Furthermore, any one monosaccharide has the potential of being linked to more than two other monosaccharides,thus generating a branch point and nonlinear polymers. Glycosidic bonds are usually formed between the growing polysaccharide chain and a covalently modified form of a monosaccharide.Such modifications include a phosphate(e.g.,glucose6-phosphate)or a nucleotide (e.g.,UDP-galactose) :

c_o-Glucopyranose

into common table sugar (Figure2-19). Larger polysaccharides,containing dozensto hundredsof

polymer of the B anomer of glucose. Human digestiveenzymescan hydrolyze the c glycosidic bonds in starch but nor the B glycosidic bonds in cellulose.Many speciesof plants,

Glucose 6-phosphate

UDp-galactose

The epimeraseenzymesthat interconvert differenr monosaccharides often do so using the nucleotide sugarsrather than the unsubstitutedsugars. Many complex polysaccharidescontain modified sugars that are covalently linked to various small groups, particularly amino, sulfate, and acetyl groups. Such modifications are abundant in glycosaminoglycans,major polysaccharide components of the extracellular matrix that we describein Chapter 19.

PhospholipidsAssociateNoncovalentlyto Form the BasicBilayerStructureof Biomembranes Biomembranes are large flexible sheets that serve as the boundaries of cells and their intracellular organelles and

cHroH H

*oi

):o

il

\?'

H

HO

> FIGURE HOH 2-19 Formationof the disaccharides lactoseand Galactose sucrose.In anyglycosidic Iinkage, theanomeric carbonof onesugar CH,OH (ineithertheo or B molecule ,f.o. conformation) islinkedto a hydroxyl oxygenon anothersugar 1)a moleculeThelinkages arenamed H O OH accordingly; thuslactose contains a B(1--+4) bond,andsucrose HOH contains an ct(1-+ 2) bond Glucose

HOH Glucose

cH2oH

\T

46

CHAPTER2 |

cHEM|CALFOUNDAT|ONS

OHH Fructose

Sucrose

Fatty acid chains

Hydrophobictail PHOSPHATIDYI. FIGURE2-20 phosphatidylcholine,a typical phosphoglyceride. havinga are amphipathicphospholipids, All phosphoglycerides hydrophobictail (yellow)and a hydrophilichead(blue)in which glycerolis linkedvia a phosphategroup to an alcohol.Eitheror both

may be saturated of the fatty acylsidechainsin a phosphoglyceride acid(red),the simplestphospholipid, or unsaturatedIn phosphatidic the phosphateis not linkedto an alcohol

form the outer surfacesof some viruses.Membranes literally define what is a cell (the outer membrane and the contents within the membrane) and what is not (the extracellular space outside the membrane). Unlike the proteins, nucleic acids, and polysaccharides,membranesare assembledby the noncoualentassociationof their componentbuilding blocks. The primary building blocks of all biomembranesare phospholipids, whose physical properties are responsiblefor the formation of the sheetlike structure of membranes.The structuresand functions of membranes,which include in addition to phospholipidsa variety of other molecules(e.g'' cholesterol,glycolipids, proteins), will be describedin detail in Chapter 10. Phospholipidsconsistof two long-chain,nonpolar fatty acid groups linked (usually by an ester bond) to small, highly polar groups, including a phosphateand a short organic molecule, such as glycerol (trihydroxy propanol) ( F i g u r e2 - 2 0 ) .

Fatty acids consist of a hydrocarbon chain attachedto a carboxyl group (-COOH)' Like glucose,fatty acids are an important energy source for many cells (Chapter 12)' They differ in length, although the predominant fatty acids in cells have an even number of carbon atoms' usually 14, 16. 18. or 20. The maior fatty acids in phospholipids are listed in Table 2-4. Fatty acids often are designatedby the abbreviationCx:y, where x is the number of carbonsin the chain and y is the number of double bonds. Fatty acids containing 12 or more carbon atoms are nearly insolublein aqueous solutions becauseof their long hydrophobic hydrocarbon chains. Fatty acids with no carbon-carbon double bonds are said to be saturated;thosewith at leastone double bond are unsaturated. Unsaturated fatty acids with more than one carbon-carbon double bond are referred to as polyunsaturated. Two "essential" polyunsaturated fatty acids, linoleic acid (C18:2)and linolenicacid (C18:3),cannotbe synthesized

(IONIZEO T()RM INPARENTHESES} ABBREVIATI()N NAME OFACII) COMMON SATURATEDFATTY ACIDS Myristic (myristate)

C14:0

cH3(cH2)12cooH

Palmitic (palmitate)

C|6:0

cH3(cH2)14cooH

Stearic (stearate)

C18:0

cH3(cH2)15cooH

Oleic (oleate)

C18:1

CH3(CH2)7CH:CH(CHz)7COOH

Linoleic (linoleate)

C18:2

CH3(CH2)4CH:CHCHzCH:CH(CHz)7COOH

Arachidonic (arachidonate)

C20:4

CH3(CH2)4(CH:CHCHz):CH:CH(CH2)3COOH

UNSATURMED FATTY ACIDS

OF CELLS B U I L D I N GB L O C K S CHEMICAL

47

by mammals and must be suppliedin their diet. Mammals can synthesizeother common fatty acids. Two stereoisomeric configurations, cis and trans, are possible around each carbon-carbondouble bond:

(seeFigure 2-20) and triacylglycerols,or triglycerides,which contain three acyl groups esterfiedto glycerol:

?

H 3 C - ( C H 2 ) , - C- O - C H 2

ol H3C-(CH2)"-C-O-CH

ol Trans

A cis double bond introducesa rigid kink in the otherwise flexible straight chain of afatty acid (Figure2-21). In general, the unsaturated fatty acids in biological systems contain only cis double bonds. Saturatedfatty acids without

solid margarine sticks) and other food producrs are not natural, arising from the catalytic processused for hydrogenation. Saturated and trans fatty acids have similar physical properties, and their consumption, relative to the consumption of unsaturatedfats, is associatedwith increasedplasma cholesterollevels. Fatty acids can be covalently attached to another molecule by a type of dehydration reaction calledesterification, in which the OH from the carboxyl group of the fatty acid and a H from a hydroxyl group on the other molecule are Iost. In the combined molecule formed by this reaction, the portion derived from the fatty acid is called an acyl group, or fatty acyl group. This is illustrated by the most common form of phospholipids, phosphoglycerides,with two acyl groups attached to rwo of the hydroxyl groups of glycerol

H3C-(CH2),-C-O-CH' Triacylglycerol Fatty acyl groups also can be covalently linked to another fatty molecule,cholesterol,to form cholesterylesters. Triglycerides and cholesteryl esters are extremely waterinsoluble compounds in which fatty acids and cholesterol are either stored or transported. Tiiglycerides are the storageform of fatty acids in the fat cells of adiposetissue and are the principal components of dietary fats. Cholesteryl estersand triglycerides are transported between tissues through the bloodstreamin specializedcarrierscalledlipoproteins(Chapter 14). In phosphoglycerides,one hydroxyl group of the glycerol is esterifiedto phosphate while the other two normally are esterified to fatty acids. The simplest phospholipid, phosphatidic acid, contains only these components. In most phospholipids found in membranes,the phosphate group is also esterified to a hydroxyl group on another hydrophilic compound. In phosphatidylcholine, for example, choline is attached to the phosphate (see Figure 2-20). The negative charge on the phosphate as well as the charged or polar groups esterifiedto it can interact strongly with water. The phosphate and its associatedesterifiedgroup, the "head" group of a phospholipid, is hydrophilic, whereas the fatty acyl chains, the "tails," are hydrophobic. (Other common phosphoglycerides and associatedheadgroupsare shown in Table2-5.) Molecules such as phospholipids that have both hydrophobic and

HHHHHHHHHHHHHH

t t-t 'qt z- c, o- _ _ c _ c _ _ _ _ _ H s -c gt t-l lgl l-l lgl-t ' g c c c c c a c- \. ^ _ t t t .HtH. .Ht H. H H Ht lHt Hl tHLHt iH U T i i U Palmitate (ionized form of palmitic acid) FfGURE2-21 The effect of a double bond on the shape of fatty acids.Shownare chemicalstructuresof the ionizedform of palmiticacid,a saturatedfatty acidwith 16 C atoms,and oleicacid, an unsaturated one with 18 C atoms In saturatedfattv acids.the

48

cHAPTER2 |

cHEMTCALFOUNDATTONS

Oleate (ionized form of oleic acid) hydrocarbonchainis often linear;the cis doublebond in oleate createsa rigid kink in the hydrocarbonchain [AfterL Stryer, 1994, Biochemistry,4th ed, W H Freeman p 265l andCompany,

GBOUP PHOSPHOGIYCERIOES HEAD COMMON c H g^ , , | ,rung

Phosphatidylcholine

r Glycosidic bonds are formed betweeneither the o or the B anomer of one sugar and a hydroxyl group on another sugar, leading to formation of disaccharidesand other (seeFigure2-L9). polysaccharides

o&N\cr. Gholine H l.H

Phosphatidylethanolamine

o&N\n Ethanolamine H lrH

Phosphatidylserine

o---rN I o4o

sine (C), thymine (T), and uracil (U) (seeFigure Z-17). A, G, ! and C are in DNA, and A, G' U, and C are in RNA. r Glucoseand other hexosescan exist in three forms: an open-chain linear structure, a six-member (pyranose) ring, and a five-member(furanose)ring (seeFigure 2-18). In biological systems,the pyranose form of l-glucose predominates.

H

Serine

Phosphatidylinositol lnositol

hydrophilic regionsare calledamphipathic.In Chapter 10, we will seehow the amphipathic properties of phospholipids are responsiblefor the assemblyof phospholipidsinto sheetlike bilayer biomembranes in which the fatty acyl tails point into the center of the sheet and the head groups point outward toward the aqueousenvironment(seeFigure2-13).a

C h e m i c a lB u i l d i n g B l o c k so f C e l l s r Three major biopolymersformed by polymerizationreactions (net dehydration)of basicchemicalbuilding blocks are presentin cells:proteins,composedof amino acidslinked by peptidebonds;nucleicacids,composedof nucleotideslinked composedof by phosphodiesterbonds;and polysaccharides, (sugars) glycosidic bonds (see linked by monosaccharides fourth major chemical the Figure 2-13). Phospholipids, into biomembranes. noncovalently assemble building block, r Differences in the size, shape, charge, hydrophobicity, and reactivity of the side chains of the 20 common amino acids determine the chemical and structural properties of proteins (seeFigure2-14). r The basesin the nucleotidescomposing DNA and RNA are carbon- and nitrogen-containingrings attached to a pentose sugar. They form two groups: the purinesadenine (A) and guanine (G)-and the pyrimidines-cyto-

r Phospholipids are amphipathic molecules with a hydrophobic tail (often two fatty acyl chains) connectedby a small organic molecule (often glycerol) to a hydrophilic head (seeFigure 2-20). The long hydrocarbon chain of a fatty acid may contain no carbon-carbon double bond (saturated) or one or more double bonds (unsaturated);a cis double bond bends the chain'

E

C h e m i c aEl q u i l i b r i u m

We now shift our discussionto chemical reactions in which bonds, primarily covalent bonds in reactant chemicals' are broken and new bonds are formed to generatereaction products. At any one time, severalhundred different kinds of chemical reactions are occurring simultaneouslyin every cell, and many chemicalscan, in principle' undergo multiple chemical reactions. Both the extent to which reactions can proceed and the rate at which they take place determine the chemical composition of cells. '$7hen reactants first mix together-before any products have been formed-their rate of reaction to form products (forward reaction)is determinedin part by their initial concentrations, which determine the likelihood of reactants bumping into one another and reacting (Figure 2-22). As the reaction products accumulate' the concentration of each reactant deir."r.r and so does the forward reaction rate. Meanwhile' some of the product molecules begin to participate in the reversereaction, which re-forms the reactants (the ability of a reaction to go "backward" is called microscopicreuersibility). This reversereaction is slow at first but speedsup as the conEventually,the ratesofthe forcentrationofproduct increases. ward and reversereactionsbecomeequal, so that the concentrations of reactantsand products stop changing.The systemis then said to be in chemical equilibrium (plural: eqwilibria). At equilibrium, the ratio of products to reactants, called the equilibrium constant' is a fixed value that is independent of the rate at which the reaction occurs. The raie of a chemical reaction can be increased by a catalyst, which acceleratesthe chemical transformation (making and breaking of covalent bonds) but is not permanently changedduring a reaction (seeSection2.4)'ln this section, we discuss several aspectsof chemical equilibria; in the next section, we examine energy changes during reactions and their relationshipto equilibria.

C H E M I C A LE Q U I L I B R I U M

49

Rate."r"rr. : 1. By rearranging theseequations,we can express the equilibrium constant as the ratio of the rate consrants Rateof forward reaction (concentrationof reactantsdecreases)

K-^

1

k1 _T

t) -1\

o

C h e m i c ael o u i l i b r i u m (forwardand reverseratesare e q u a l ,n o c h a n g ei n c o n c e n t r a t i o n of reactantsand products)

; E o

Rate of reverse reaction (concentration of products increases)

When reactantsare first mixed. initial concentrationof products= 0

T i m e+ A FIGURE 2-22 Timedependence of the ratesof a chemical reaction.Theforwardandreverse ratesof a reaction dependin oart on the initialconcentrations of reactants andproducts. Thenet forwardreaction rateslowsasthe concentration of reactants decreases, whereas the netreverse reaction rateincreases asrne concentration of products increases At equilibrium, the ratesof the forwardandreverse reactions areequalandthe concentrations of reactants andproducts remainconstant

E q u i l i b r i u mC o n s t a n t sR e f l e c t h e Extent of a ChemicalReaction The equilibrium constant K.o dependson the nature of the reactants and products, the temperature, and the pressure (particularly in reacrions involving gases).Under standard physicalconditions (25'C and 1 atm pressurefor biological systems),the K.o is always the same for a given reaction, whether or not a catalystis present. For the general reaction with three reactants and three products aA + bB * cC ;

. zZ + yY + xX

(2_1)

where capital letters representparticular moleculesor atoms and lowercaseletters representthe number of each in the reaction formula, the equilibrium constant is given by

(2-2) where bracketsdenote the concentrationsof the moleculesat equilibrium. The rate of the forward reaction (left to right in Equation (2-1) is R a r e 6 o , * , ,:6 k r l A l " l B l b [ C | . where Ai is the rate constant for the forward reaction. Similarly, the rate of the reversereaction (right to left in Equation 2-1) is : k,[X]" [y lv Ratereverse [Z]" where A, is the rate constant for the reversereaction. At equilibrium the forward and reverserates are equal, so Rateso*..6/

s0

CHAPTER2 I

CHEMICALFOUNDATIONS

ChemicalReactionsin CellsAre at SteadyState Under appropriate conditions and given sufficient time, individual biochemical reactions carried out in a test tube eventually will reach equilibrium. Within cells, however, many reactions are linked in pathways in which a product of one reaction servesas a reactarftin another or is pumoed out of the cell. In this more complex situation,when the iate of formation of a substanceis equal to the rate of its consumption, the concentration of the substanceremains constant, and the systemof linked reactionsfor producing and consuming that substanceis said to be in a steady state (Figure 2-23). One consequenceof such linked reactionsis that they prevent the accumulation of excessintermediates,protecting cells from the harmful effects of intermediatesthat have the Dotential of beingtoxic at high concentrations.

DissociationConstantsof Binding Reactions Reflectthe Affinity of lnteractingMolecules The concept of equilibrium also appliesto the binding of one molecule to another. Many important cellular processesdepend on such binding "reactions," which involve the making and breaking of various noncovalentinteractionsrather than covalentbonds,as discussedabove.A common exampleis the binding of a ligand (e.g.,the hormone insulin or adrenaline)to its receptor on the surface of a cell forming a multimolecular assembly,or complex, that triggersa biological response.Another exampleis the binding of a protein to a specificsequence of basepairs in a moleculeof DNA, which frequently causes the expressionof a nearby geneto increaseor decrease(Chapter 7). lf the equilibrium constant for a binding reaction is (a)Testtube equilibriumconcentrations AAA

=

-BBB

BBB BBB

(b) Intracellularsteady-stateconcentrations n n - B B_B

BBB

_

- cC CC

A FIGURE2-23 Comparisonof reactionsat equilibrium and steady state. (a) ln the test tube, a biochemicalreaction(A -+ B) eventuallywill reachequilibrium,in which the ratesof the fon,rardand reversereactionsare equal(asindicatedby the reactionarrowsof equal length).(b) In metabolicpathwayswithin cells,the productB commonly would be consumed,in this exampleby conversionto C. A pathwayof linkedreactionsis at steadystatewhen the rateof formationof the (e g , B) equalstheir rateof consumption.As indicatedby intermediates the unequallengthof the arrows,the individualreversible reactions constitutinga metabolicpathwaydo not reachequilibrium Moreover, the concentrations of the intermedrates at steadystatecan differfrom what theywould be at eouilibrium.

CanBind MultipleLigands ffi Rodcast:Macromolecules > FIGURE 2-24 Macromolecules can havedistinctbinding M u l t i l i g a n db i n d i n gm a c r o m o l e c u l (ee . 9 . ,p r o t e i n ) (eg , a protein, sitesfor multipleligands.A largemacromolecule Bindingsite A (K6a) bindingsites(A-C)isshown;eachbinding B i n d i n gs i t e B ( K 6 s ) blue)withthreedistinct molecular to threedifferent binding complementarity siteexhibits (ligands (KdAc) partners A-C)with distinct dissociation constants L i g a n dB

known, the intracellular stability of the resultingcomplex can ( e . 9 . s, m a l l be predicted.To illustrate the generalapproach for determin- m o l e c u l e ) ing the concentrationof noncovalentlyassociatedcomplexes, we will calculatethe extent to which a protein (P) is bound to DNA (D)forming a protein-DNA complex (PD):

L i g a n dA ( e . 9 . s, m a l l p r o t e i n )

P+D.-PD Most commonly, binding reactions are describedin terms of the dissociation constant K6, which is the reciprocal of the equilibrium constant. For this binding reaction, the dissociation constant is given by

ro:

tPIIDl

TDi

L i g a n dC (e 9.,polysaccharide)

16:\ Binding s i t eC ( K 6 6 ) d

(2-4)

Typical reactionsin which a protein binds to a specificDNA sequencehavea K6 of 10-10 M, where M symbolizesmolariry or moles per liter (mol/L). To relate the magnitude of this dissociation constant to the intracellular ratio of bound to unbound DNA, let's consider the simple example of a bacterial cell hav1s L ing a volume of 1.5 x 10 and containing L moleculeof DNA and 10 moleculesof the DNA-binding protein P. In this 0 case,givena Ka of 10-1 M, 99 percentof the time this specific DNA sequencewill have a molecule of protein bound to it and 1 percent of the time it will not, even though the cell contains only 10 moleculesof the protein! Clearly P and D bind very tightly (have a high affinity), as reflectedby the low value of the dissociation constant for their binding reaction. For protein' M protein and protein-DNA binding, K6 values of <10 o -10 (nanomolar)are consideredto be tight, M (micromolar) modestlytight, and -10-' M (millimolar) relativelyweak. The large size of biological macromolecules,such as proteins, can result in the availability of multiple surfacesfor complementaryintermolecularinteractions (Figure 2-24). As a consequence,many macromoleculeshave the capacity to bind severalother moleculessimultaneously.In some cases, these binding reactionsare independent,with their own distinct Ka valuesthat are constant.In other cases,binding of a moleculeat one site on a macromoleculecan changethe threedimensionalshapeof a distant site, thus altering the binding interactionsof that distant sitewith someother molecule.This is an important mechanismby which one moleculecan alter (regulate)the activity of a secondmolecule(e.g.,a protein) by changingits capacityto interact with a third molecule.'Weexamine this regulatory mechanismin more detail in Chapter 3. RoshanKetab 021-6 69 50 639

B i o l o g i c aFl l u i d sH a v eC h a r a c t e r i s t ipcH V a l u e s The solventinsidecellsand in all extracellularfluids is water. An important characteristic of any aqueous solution is the

concentrationof positivelychargedhydrogenions (H+ ) and negatively chargedhydroxyl ions (OH-). Becausetheseions are the dissociationproducts of H2O, they are constituentsof all living systems,and they are liberatedby many reactionsthat take place between organic moleculeswithin cells.These ions also can be transported into or out of cells, as when highly acidic gastric juice is secretedby cellslining the walls of the stomach. \fhen a water moleculedissociates,one of its polar H-O bonds breaks.The resultinghydrogenion, often referredto as a proton, has a short lifetime as a free ion and quickly combines with a water molecule to form a hydronium ion (HrO*). For convenience,however'we refer to the concentration of hydrogenions in a solution, [H*], eventhough this really representsthe concentrationof hydronium ions, [H3O-]. Dissociationof H2O generatesone OH- ion along with each H*. The dissociationof water is a reversiblereaction: H2O:H*+OHAt 25 'C, [H+][OH-]_:

1O-r4 M2, so that in pure water,

l H * l: t o H - l : 1 o - ' M .

The concentration of hydrogen ions in a solution is expressedconventionally as its pH, defined as the negativelog of the hydrogen ion concentration. The pH of pure water at 25 'C is 7:

p H : - l o g [ H -:] l " s E L : b e + = - : z lnj,J

It is important to keep in mind that a 1 unit differencein pH representsa tenfold difference in the concentration of protons. On the pH scale,7.0 is consideredneutral: pH values below 7.0 indicateacidicsolutions(higher[H*]), and values above 7.0 indicate basic (alkaline) solutions (Figure 2-25)' For instance, gastric juice, which is rich in hydrochloric acid EQUILIBRIUM CHEMICAL

51

I n c r e a s i n g lbya s i c ( l o w e rH +c o n c e n t r a t i o n )

pH scale

1 4 S o d i u mh y d r o x i d(e1 N ) 13 1)

H o u s e h o lb dl e a c h A m m o n i a( 1 N )

11

1 0 1^

sea water , _.1r I n t e r i o ro f c e l l Fertilizedegg U n f e r t i l i z eedg g

8 j' 6

Urine I n t e r i o ro f t h e l y s o s o m e G r a p e f r u ijtu i c e

ric acid (HCl) or the carboxylgroup (-COOH), which tends to dissociateto form the negativelychargedcarboxylate ion (-COO ). Likewise,a baseis any molecule,ion, or chemical group that readily combineswith a H+, such as the hydroxyl ion (OH-); ammonia (NH:), which forms an ammonium ion (NH+-); or the amino group (-NH2). rWhenacid is addedto an aqueoussolution,the [H+] increases(the pH goes down). ConverselS when a base is addedto a solution,the [H+] decreases (the pH goesup). Because[H+][OH ] : 10-14M2, any increasein [H*] is coupled with a commensuratedecrease in [OH-] and vice versa. Many biologicalmoleculesconrain both acidic and basic groups.For example,in neutral solutions(pH : 7.0), many amino acidsexist predominantlyin the doubly ionizedform, in which the carboxyl group has lost a proton and the amino group has acceptedone: NH,*

t-

H-C-COOR

G a s t r i cj u i c e

where R representsthe uncharged side chain. Such a molecule, containing an equal number of positive and negative ions, is called a zwitterion. Zwitterions, having no net charge,are neutral. At extreme pH values,only one of these two ionizable groups of an amino acid will be charged. The dissociationreaction for an acid (or acid group in a I n c r e a s i n gal cy i d i c larger molecule)HA can be wrirten as HA == H+ + A . The ( gr e a t eH r +c o n c e n t r a t i o n ) equilibriumconstantfor this reaction,denotedK. (thesubscript FfGURE2-25 pH valuesof commonsolutions.ThepHof an d standsfor "acid"), is definedas K" : [H*][A ]/lHAl. Taking aqueous solution isthenegative logof thehydrogen ionconcentratron the logarithm of both sidesand rearrangingthe result yields a ThepHvalues for mostintracellular andextracellular biological fluids very usefulrelation betweenthe equilibrium constantand pH: arenear7 andarecarefully regulated to permittheproperfunctioning of cells, organelles, andcellular secretions H y d r o c h l o r i ac c i d ( 1 N )

(HCl), has a pH of about 1. ks [H+] is roughly a millionfold greaterthan that of cytoplasmwith a pH of abort 7.2. Although the cytosolof cellsnormally has a pH of about 7.2, the pH is much lower (about 4.5) in the interior of lyso, somes,one type of organellein eukaryoticcells(Chapter9). The many degradative enzymeswithin lysosomesfunction optimally in an acidic environment,whereastheir action is inhibited in the near neurral environmentof the cytoplasm. This illustratesthat mainrenanceof a specificpH is essential for proper functioning of some cellular strucrures.On the other hand, dramatic shifts in cellular pH may play an important role in controlling cellularactivity.For example,the pH of the cytoplasmof an unfertilizedeggof the seaurchin, an aquatic animal, is 6.6. Within 1 minute of fertilization, however,the pH risesto 7.2; that is, the [H+l decreases to about one-fourth its original value, a change necessaryfor subsequentgrowth and division of the egg.

Hydrogenlons Are Releasedby A c i d sa n d T a k e nU p b y B a s e s In general, an acid is any molecule, ion, or chemical group that tendsto releasea hydrogenion (H+), suchas hydrochlo52

.

c H A p r E R 2| c H E M t c A L F o u N D A T t o N S

pH: pK"+ loe#l

lr1Al

e-s)

where pK" equals-log K". From this expression,commonly known astheHendersonHasselbaLch equation,it can be seenthar the pK. of any acid is equalto the pH at which half the moleculesare dissociatedand half are neutral (undissociated). This is becausewhen [A-] : [HA], then log ([A-]/[HA]) : O, and thus pK" : pH. The Henderson-Hasselbalch equarionallows us to calculatethe degree of dissociationof an acid if both the pH of the solution and the pK, of the acid are known. Experimentally,by meas, uring the [A ] and [HA] as a function of the solution'spH, one can calculatethe pK. of the acid and thus the equilibrium constant Ka for the dissociationreacion (Figure2-26).

B u f f e r sM a i n t a i nt h e p H o f I n t r a c e l l u l a r a n d E x t r a c e l l u l aFr l u i d s A growing cell must maintain a consrant pH in the cytoplasm of about 7.2-7.4 despitethe metabolic production of many acids,suchas lactic acid and carbon dioxide;the latter reactswith water to form carbonicacid (H2CO3).Cellshave a reservoir of weak basesand weak acids, called buffers, which ensure that the cell's pH remains relatively constant

H2CO3:HCO3-+H* H2C03 -';i9 1 1 0 0

= =)

.oO

6P OG tsc

-o

50

0L

0

7.48 OH

2-26 lhe relationshipbetween pH, pKa,and the A FfGURE of carbonic acid of an acid.Asthe oHof a solution dissociation in the of the compound risesfrom0 to 8 5, the percentage from100 form(HzCO:) decreases or un-ionized, undissociated, from0 percentWhen percent formincreases andthatof the ionized acidhas the pH(6.4)isequalto the acidspKa,halfof the carbonic allof the acidhas ionizedWhenthe pH risesto above8, virtually form(HCOr) ionized to the bicarbonate despitesmall fluctuationsin the amountsof H* or OH- being generatedby metabolism or by the uptake or secretionof moleculesand ions by the cell. Buffers do this by "soaking up" excessH* or OH when theseions are addedto the cell or are producedby metabolism. If additional acid (or base)is added to a buffered solution whose pH is equal to the pK, of the buffer (tHAl : [A ]), the pH of the solution changes,but it changesless than it would if the buffer had not been present.This is becauseprotons releasedby the addedacid are taken up by the ionized form of the buffer (A- ); likewise, hydroxyl ions gener:atedby the addition of baseare neutralized by protons releasedby the undissociatedbuffer (HA). The capacity of a substanceto releasehydrogen ions or take them up depends partly on the extent to which the substancehas already taken up or releasedprotons, which in turn dependson the pH of the solution relative to the pK" of the substance.The ability of a buffer to minimize changesin pH, its buffering capacity,dependson the concentration of the buffer and the relationship between its pK" value and the pH, which is exequation. pressedby the Henderson-Hasselbalch The titration curve for acetic acid shown inFigure 2-27 illustratesthe effect of pH on the fraction of moleculesin the un-ionized(HA) and ionizedforms (A ). At one pH unit below the pK" of an acid, 91 percentof the moleculesare in the HA form; at one pH unit above the pK^, 9L percent are in the A form. At pH values more than one unit above or below the pK", the buffering capacity of weak acids and basesdeclines rapidly. In other words, the addition of the same number of moles of acid to a solution containing a mixture of HA and A that is at a pH near the pK" will cause lessof a pH changethan it would if the HA and A- were not present or if the pH were far from the pK. value. All biological systemscontain one or more buffers. Phosphate ions, the ionized forms of phosphoric acid, are present in considerablequantitiesin cellsand are an important factor

o

0'8 0.6 0.4 0.2 CH3COOH of dissociated Fraction AddedOH- -----)

1.0

2-27 The titration curveof the buffer aceticacid FfGURE of aceticacidto hydrogen (CH3COOH). ThepK,for the dissocration are ionsis4 75 At thispH,halftheacidmolecules andacetate thesolution scale, pHismeasured on a logarithmic Because dissociated CH3COOH at pH3.75to 9 percent CH3COOH from91 percent changes pH range in this capacity buffering maximum has at pH5 75.Theacid in maintaining, or buffering, the pH of the cytoplasm. Phosphoric acid (H3POa) has three protons that are capable of dissociating,but they do not dissociatesimultaneously.Loss of each proton can be describedby a discretedissociationreaction and pKo, as shown in Figure 2-28.The titration curve 14

P K a =1 2 ' 7 HPO42-+

12

I

PKa=7'2

H2PO4-*

o_

PKa=2'1

PO4

HPO+2-+H*

H3PO4+HrPOf+H+

Added OH- -----> A FIGURE2-28 The titration curve of phosphoric acid (H3PO4), ubiquitous a common buffer in biological systems.Thisbiologically at differentpH moleculehasthreehydrogenatomsthat dissociate acidhasthreepK" values,as notedon the values;thus phosphoric graph The shadedareasdenotethe pH ranges-withinone pH unit of the three pKuvalues-where the bufferingcapacityof phosphoric acidis high In theseregions,the additionof acid(or base)will cause smallchangesin the PH relatively EQUILIBRIUM CHEMICAL

53

for phosphoric acid shows that the pK" for the dissociationof the secondproton is 7.2. Thus at pH7.2, about 50 percentof cellula^rphosphate is H2POa- and about 50 percent is HPO4'- according to the Henderson-Hasselbalchequation. For this reason,phosphateis an excellentbuffer at pH values around 7.2, the approximate pH of the cytoplasm of cells, and at pH 7.4, the pH of human blood.

C h e m i c a lE q u i l i b r i u m r A chemical reaction is at equilibrium when the rate of the forward reaction is equal to the rate of the reversereaction (no net changein the concentration of the reactants or products). The equilibrium constant K.o of a reaction reflects the tio of products to reacrantsai equilibrium and thus is a measure of the extent of the reaction and the relative stabilities of the reactantsand products. r The K.o depends on the temperature, pressure, and chemical properties of the reactants and products but is independent of the reaction rate and of the initial concentrations of reactantsand products. For any reaction, the equilibrium constant K.o equalsthe tio of the forward rate constant to the reversi rate constant (krl&r).The rates of conversion of reactantsto products and vice versa depend on the rate constants and the concentrationsof the reactantsor products. 'Sfithin r cells, the linked reactions in metabolic pathways generallyare at steadystate,not equilibrium, at which rate of formation of the intermediatesequals their rate of consumption (seeFigure 2-23) and thus the concentrations of the intermediatesare not changing. r The dissociation constant K6 for the noncovalent binding of two molecules is a measure of the stability of the complex formed betweenthe molecules(e.g.,ligand-receptor or protein-DNA complexes). r The pH is the negativelogarithm of the concentration of hydrogen ions (-log [H+]). The pH of the cytoplasm is normally about 7.2-7.4, whereasthe interior of lysosomeshas a pH of about 4.5. r Acids releaseprotons (H+) and basesbind them. In biological molecules,the carboxyl and phosphate groups are the most common acidic groups; the amino group is the most common basicgroup. r Buffers are mixrures of a weak acid (HA) and its corresponding baseform (A ), which minimize the changein pH of a solution when acid or alkali is added. Biological systems use various buffers to maintain their pH within a very narrow range.

Biochemical Energetics

W

The production of energy,its storage,and its use are central to the economy of the cell. Energy may be defined as the ability to do work, a concept applicable to automobile 54

.

c H A p r E R 2| c H E M t c A L F o u N D A T t o N s

engines and electric power plants in our day-to-day world and to cellular enginesin the biological world. The energy associatedwith chemical bonds can be harnessedto support chemical work and the physical movements of cells.

SeveralFormsof EnergyAre lmportant i n B i o l o g i c aS l ystems There are two principal forms of energy:kinetic and potential. Kinetic energyis the energyof movement-the motion of molecules,for example.The secondform of energy,potential energy, or stored energy, is particularly important in the study of biological or chemical systems. Thermal energy, or heat, is a form of kinetic energy-the energy of the motion of molecules.For heat to do work, it must flow from a region of higher temperature-where the averagespeedof molecularmotion is greater-to one of lower temperature. Although differencesin temperature can exist betweenthe internal and external environmentsof cells,these thermal gradientsdo not usually serveas the sourceof energy for cellular activities.The thermal energy in warm-blooded animals, which have evolveda mechanismfor thermoregulation, is usedchiefly to maintain constantorganismictemperatures. This is an important function becausethe rates of many cellular activities are temperature-dependent.For example, cooling mammalian cellsfrom their normal body temperature of 37 "C to 4 "C can virtually "freeze" or stop many cellular processes(e.g.,intracellularmembranemovements). Radiant energyis the kinetic energyof photons, or waves of light, and is critical to biology. Radiant energycan be converted to thermal energy,for instancewhen light is absorbed by moleculesand the energy is converted to molecular motion. Radiant energy absorbed by moleculescan also change the electronic structure of the molecules, moving electrons into higher-energystates (orbitals), whence it can later be recoveredto perform work. For example, during photosynthesis,light energy absorbed by specializedmolecules (e.g.,chlorophyll) is subsequentlyconverted into the energy of chemical bonds (Chapter 12). Mechanical energy,a major form of kinetic energy in biology usually resultsfrom the conversionof stored chemical energy.For example, changesin the lengths of cytoskeletal filaments generate forces that push or pull on membranes and organelles(Chapters 17 and 18). Electric energy-the energy of moving electrons or other charged particles-is yet another major form of kinetic energy. Several forms of potential energy are biologically significant. Central to biology is chemicalpotential energy,the energy stored in the bonds connecting atoms in molecules.Indeed, most of the biochemicalreactionsdescribedin this book involve the making or breaking of at least one covalent chemical 'We bond. recognize this energy when chemicals undergo energy-releasingreactions. For example, the high potential energy in the covalent bonds of glucosecan be releasedby controlled enzymaticcombustion in cells (Chapter 12). This energyis harnessedby the cell to do many kinds of work. A secondbiologically important form of potential energyis the energyin a concentration gradient. \X/henthe concentration

of a substanceon one side of a barrier,such as a membrane,is different from that on the other side, a concentration gradient exists. All cells form concentration gradients between their interior and the external fluids by selectivelyexchangingnutrients, waste products, and ions with their surroundings.Also, organelleswithin cells (e.g., mitochondria, lysosomes)frequently contain different concentrationsof ions and other molecules;the concentration of protons within a lysosome, as we saw in the last section,is about 500 timesthat of the cytoplasm. A third form of potential energy in cells is an electric potential-the energy of charge separation. For instance, there is a gradient of electric charge of =200,000 volts per cm acrossthe plasma membrane of virtually all cells.We discuss how concentration gradients and the potential differenceacrosscell membranesare generatedand maintained in Chapter 11 and how they are converted to chemical potential energyin Chapter 12.

CellsCanTransformOne Type of Energyinto Another According to the first law of thermodynamics,energy is neither creatednor destroyedbut can be converted from one form to another.(In nuclear reactions,massis convertedto energy, but this is irrelevantto biological systems.)In photosynthesis, for example,the radiant energyof light is transformedinto the chemicalpotential energy of the covalent bonds betweenthe atoms in a sucroseor starch molecule.In musclesand nerves, chemical potential energy stored in covalent bonds is transinto the kinetic energyof musclecontracformed, respectively, tion and the electricenergyof nerve transmission.In all cells, potential energy,releasedby breaking certain chemicalbonds, is used to generatepotential energyin the form of concentration and electricpotential gradients.Similarly,energystoredin chemical concentration gradients or electric potential gradients is usedto synthesizechemicalbonds or to transport moleculesfrom one side of a membraneto another to generatea concentrationgradient. The latter processoccurs during the transport of nutrients such as glucoseinto certain cells and transport of many wasteproducts out of cells. Becauseall forms of energyare interconvertible,they can be expressedin the sameunits of measurement.Although the standard unit of energy is the joule, biochemistshave traditionally used an alternative unit, the calorie (1 joule : 0.239 calorie). Throughout this book, we use the kilocalorie to measureenergychanges(1 kcal : 1000 cal).

The Changein FreeEnergyDetermines the Directionof a ChemicalReaction Becausebiological systemsare generally held at constant temperatureand pressure,it is possibleto predict the direction of a chemicalreaction from the changein the free energy G, named after J. V. Gibbs, who showed that "all systems changein such a way that free energy [G] is minimized." In products, the the caseof a chemical reaction, reactants * free-energychangeAG is given by AG

:

Goroaucts -

Greactants

(a) Endergonic

1

A I

I

(\

ah

t 6

j o

c) 0)

o) (!) 0) LI

E

u

Reactants

Progressof reaction----->

Progressof reaction----->

2-29 Changesin the free energy(AG)of exergonic FIGURE the freeenergy reactions, and endergonicreactions.(a)In exergonic Consequently, islowerthanthatof the reactants of the products asthe andenergyisreleased occurspontaneously thesereactions reactions, the freeenergyof the proceed(b)In endergonic reactions do andthesereactions productsisgreaterthanthat of the reactants of energymustbe source An external notoccurspontaneously into products areto be converted suppliedif the reactants The relation of AG to the direction of any chemical reaction can be summarizedin three statements: r If AG is negative,the forward reaction will tend to occur spontaneouslyand energy usually will be releasedas the reaction takes place (exergonicreaction) (Figure2-291. r If AG is positive, the forward reaction will not occur spontaneously:energywill have to be added to the systemin order to force the reactantsto becomeproducts (endergonic reaction). r If AG is zero, both forward and reversereactions occur at equal rates and there will be no spontaneousconversion of reactantsto products (or vice versa);the systemis at equilibrium. By convention, the standard free-energychange of a reaction AGo' is the value of the change in free energy under the conditionsof 298 K (25 "C)' 1 atm pressure,pH 7.0 (as in pure water), and initial concentrationsof 1'M for all reacproJucts exceptprotons' which are kept at 10-7 M t"nts "nd (pH 7.0). Most biological reactions differ from standard conditions, particularly in the concentrations of reactants' which are normally lessthan 1 M. The free energy of a chemical system can be defined as : H - TS, where H is the bond energy'or enthalpy' of the G system; 7 is its temperature in degreesKelvin (K); and S is the entropy, a measureof its randomnessor disorder' If temperature remains constant' a reaction proceeds spontaneously only if the free-energychange AG in the following equation is negative:

AG: AH- TAS B I O C H E M I C AELN E R G E T I C S .

()-6\

55

In an exothermic reaction, the products contain less bond energy than the reactanrs, the liberated energy is usually converted to heat (the energy of molecular motion), and AH is negative.In an endothermic reaction, the products contain more bond energy than the reactants,heat is absorbed during the reaction, and AH is positive. The combined effects of the changes in the enthalpy and entropy determine if the AG for a reaction is positive or negarive. An exothermic reaction (AH < 0) in which enrropy increases(AS > 0) occurs spontaneously(AG < 0). An endothermic reaction (AH > 0) will occur spontaneouslyif AS increasesenough so that the T AS term can overcome the positive AH. Many biological reactions lead to an increase in order and thus a decreasein entropy (AS < 0). An obvious example is the reaction that links amino acids to form a protein. A solution of protein molecules has a lower entropy than does a solution of the same amino acids unlinked because the free movementof any amino acid in a protein is restricted when it is bound into a long chain. Often cells compensate for decreases in entropy by "coupling" suchsynthetic,entropylowering reactions with independent reactions that have a very highly negariveAG (seebelow). In this way cells can convert sources of energy in their environment into the building of highly organized structures and metabolic pathways that are essentialfor life. The actual changein free energy AG during a reacrion is influenced by temperature, pressure,and the initial concentrations of reactants and products and usually differs from AG''. Most biologicalreactions-like othersthat take place in aqueoussolutions-also are affected by the pH of the so'We lurion. can estimate free-energy changes for different temperaturesand initial concentrationsusing the equation AG:

AC.'-r RTln Q:

AC"'+ RTln

fproducts] Q-7) I reacrantsl

where R is the gas constanrof 1.987 call(degree.mol), T is the temperature (in degreesKelvin), and Q is the initial rctio of products to reactants. For a reaction A + B == C. in which two molecules combine to form a third, e in Equation2-7 equalstcl/lAltBl.In this case,an increasein the initial concentration of either [A] or [B] will result in a larger negative value for AG and thus drive the reaction toward more formation of C. Regardlessof the AG"' for a particular biochemicalreaction, it will proceed spontaneouslywithin cells only if AG is negative, given the intracellular concentrations of reactants and products. For example, the conversion of glyceraldehyde 3-phosphate (G3P) to dihydroxyacetonephosphate (DHAP), two inrermediatesin the breakdownof glucose, .^DHAP G3P . has a AGo' of -1840 caVmol.If the initial concentrationsof G3P and DHAP are equal, then AG : AGo' becauseRT ln 1 : 0; in this situation, the reversiblereaction G3P : DHAP will proceedspontaneouslyin the direction of DHAp formation until equilibrium is reached. However, if the initial 56

CHAPTER2 I

CHEMICALFOUNDATIONS

[DHAP] is 0.1 M and the initial [G3P] is 0.001 M, with other conditions standard,then Q in Equation 2-7 equals0.1/0.001 : 100, giving a AG of *887 caVmol.Under theseconditions, the reaction will proceed in the direction of formation of G3P. The AG for a reaction is independent of the reaction rare. Indeed, under usual physiological conditions, few if any of the biochemical reactions neededto sustain life would occur without some mechanism for increasing reaction rates. As we describe below and in more detail in Chapter 3, the rates of reactions in biological systemsare usually determinedby the activity of enzymes,the protein catalyststhat acceleratethe formation of products from reactantswithout altering the value of AG.

The Ad' of a ReactionCan Be Galculatedfrom lts K.o A chemical mixture at equilibrium is in a stable state of minimal free energy.For a system at equilibrium (AG : 0, Q : K"o), we can write AGo': -2.3RTlogK.o: -1362logK.o

Q-8)

under standard conditions (note the changeto base 10 logarithms). Thus if we determinethe concentrationsof reactants and products at equilibrium (i.e., the K.o), we can calculate the value of AGo'. For example,the K.o for the inrerconversion of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate (G3P : DHAP) is 22.2 under standard conditions. Substitutingthis value into Equation 2-8, we can easily calculatethe AGo' for this reaction as - 1840 callmol. By rearranging Equation 2-8 and taking the antilogarithm, we obtain Keq :

10-(aG"'/2'3RT)

(2-e)

From this expression,it is clear that if AGo' is negative,the exponent will be positive and henceK.o will be greater than 1. Therefore at equilibrium rhere will be more products than reactants; in other words, the formation of products from reactants is favored. Conversely,if AG'' is positive, the exponent will be negativeand K.o will be lessthan 1.

The Rateof a ReactionDependson the Activation EnergyNecessary to Energize the Reactantsinto a TransitionState As a chemical reaction proceeds, reactants approach each otherl some bonds begin to form while others begin to break. One way to think of the state of the moleculesduring this transition is that there are strains in the electronic configurations of the atoms and their bonds. In order for the collection of atoms to move from the relatively stable state of the reactants to this intermediate state during the reaction, an introduction of energy is necessary.This is illustrated in the reaction energy diagram in Figure 2-30. Thus the collection of atoms is transiently in a higher-energysrare at some point during the course of the reaction. The state during a chemicalreaction at which the systemis at its highest energy level is called the transition state or transition-

+

II

Transitionstate (uncatalyzed)

Transitionstate (catalyzed) 6 0) 0) 0) LI

Products

Progressof reaction----> 2-30 Activationenergy of uncatalyzedand A FIGURE reactionpathway catalyzedchemicalreactions.Thishypothetical (blue)depicts proceeds A thechanges in freeenergyG asa reaction reaction willtakeplacespontaneously if thefreeenergy(G)of the (AG< 0). However, products all islessthanthatof the reactants proceed reactions throughone(shownhere)or morehighchemical isinversely states, andthe rateof a reaction energytransition proportional in to theactivation energy(AG*),whichisthe difference the reactants andthetransition stateIn a freeenergybetween (red),thefreeenergies of the reactants and reaction catalyzed products stateis areunchanged but thefreeenergyof thetransition lowered, thusincreasing thevelocity of the reaction

state intermediate.The energy neededto excite the reactants to this higher-energystate is called the activation energy of the reaction. The activation energy is usually representedby AGt, analogousto the representationof the changein Gibbs free energy (AG) already discussed.From the transition state, the collection of atoms can either releaseenergy as the reaction products are formed or releaseenergy as the atoms go "backward" and re-form the original reactants.The velocity (V) at which products are generated from reactants during the reaction under a given set of conditions (temperature, pressure,reactant concentrations)will depend on the concentration of material in the transition state, which in turn will depend on the activation energy and the characteristic rate constant (z) at which the transition state ls converted to products. The higher the activation energy, the lower the fraction of reactantsthat reach the transition state and the slower the overall rate of the reaction. The relationship between the concentration of reactants,z and V is (AG+/2 3RT) v : u lreactants]x 10 From this equation, we can seethat lowering the activation energy-that is, decreasingthe free energy of the transition stateAG+-leads to an accelerationof the overall reactionrate V. A reduction in AGt of 1.36 kcal/mol leads to a tenfold increasein the rate of the reaction, whereas a 2.72 kcal/mol reduction increasesthe rate 100-fold. Thus relatively small changesin AGt can lead to large changesin the overall rate of the reaction.

Catalystssuch as enzymes(Chapter 3) acceleratereaction rates by lowering the relative energy of the transition state and so the activation energy (seeFigure 2-30). The relative energiesof reactantsand products will determineif a reaction is thermodynamically favorable (negativeAG), whereas the activation energy will determine how rapidly products form (reaction kinetics). Thermodynamically favorable reactions will not occur if the activation energiesare too high.

L i f e D e p e n d so n t h e C o u p l i n go f U n f a v o r a b l e s ith Energetically C h e m i c aR l e a c t i o nw FavorableReactions unfavorable(AG > 0) Many processesin cellsareenergetically include the synExamples and will not proceed spontaneously. of a substance transport nucleotides and thesis of DNA from a higher concenlower to from a plasma membrane acrossthe or endergonic, energy-requiring, out an can carry tration. Cells reaction (AGr > 0) by coupling it to an energy-releasing,or exergonic, reaction (AGz < 0) if the sum of the two reactionshas an overall net negativeAG. Suppose,for example,that the reactionA = B + X has a AG of + 5 kcaUmoland that the reactionX = Y + Zhas a L,G of -10 kcal/mol:

(1) A

B+X

AG:+Skcal/mol

( 2\ x

Y+Z

AG:-10kcal/mol

Sum: A.

^B+Y+Z

AG'' :-5kcal/mol

In the absenceof the secondreaction' there would be much more A than B at equilibrium. However, becausethe conversion of X to Y + Z is such a favorable reaction, it will pull the first process toward the formation of B and the consumption of A. Energetically unfavorable reactions in cells often are coupled to the energy-releasinghydrolysis of ATP' as we discussnext.

Hydrolysisof ATPReleasesSubstantialFree E n e r g ya n d D r i v e sM a n y C e l l u l a rP r o c e s s e s In almost all organisms, adenosinetriphosphate, or AIP, is the most important molecule for capturing' transiently storing, and subsequentlytransferring energy to perform work (e.g.,biosynthesis,mechanicalmotion). The useful energyin an ATP molecule is contained in phosphoanhydride bonds, which are covalent bonds formed from the condensationof two moleculesof phosphateby the loss of water: OO

ti o- -i-on + Ho-P-o- . * ll

o-

o-

o

llll o--P-O-?-OI I o o-

+ H2O

B I O C H E M I C AELN E R G E T I C S

57

pH. During synthesis of ATP, a large input of energy is required to force the negative charges in ADP and P1 together.ConverselSmuch energy is releasedwhen ATP is hy*zc'-a-N rtl \u Phosphoanhydride bonds drolyzed to ADP and P;. In comparison, formation of the HC__*,rC_;1 phosphoester o bond between an unchargedhydroxyl in glycto to - o - P -i ol |- lL|Pl - O _ | . P - O erol and P1requires less energy, and less energy is released cH2-owhen this bond is hydrolyzed. tll oo-o H Cells have evolved protein-mediated mechanismsfor transferringthe free energyreleasedby hydrolysis of phosphoanhydride bonds to other molecules, thereby driving HO OH reactions that would otherwise be energeticallyunfavorAdenosine triphosphate able. For example, if the AG for the reaction B + C -+ D is positive but less than the AG for hydrolysis of ATP, the reA FIGURE 2-31 Adenosinetriphosphate(ATP).Thetwo phosphoanhydride bonds(red)in ATP, whichlinkthethreephosphate action can be driven to the right by coupling it to hydrolygroups, eachhasa AG'of about-7 3 kcal/mol for hydrolysis sis of the terminal phosphoanhydride bond in ATP. In one Hydrolysis of thesebonds,especially theterminal one,rsrnesource common mechanism of such energy coupling, some of the of energythatdrivesmanyenergy-requiring reactions in biological energy stored in this phosphoanhydride bond is transferred systems. to one of the reactants by breaking the bond in ATP and forming a covalent bond between the releasedphosphate group and one of the reactants.The phosphorylatedinterAn ATP molecule has two key phosphoanhydride (also mediate generatedin this way can then react with C to calledphosphodiester)bonds (Figure 2-31). Hydrolysis of a form D * P; in a reaction that has a negativeAG: phosphoanhydride bond (-) in each of the following reactions has a highly negativeAG'' of about -7.3 kcal/mol: NH"

B+ATPTB-p+ADP Ap-p-p + H2O ----+ Ap-p + Pi + H+ (ATP) Ap-p-p+H2O(ATP)

B-p+C-+D+P;

(ADP) Ap+PPi+H+ (AMP)

The overallreaction B+C+ATP:-D+ADP+P.

Ap-p + H2O ----+ Ap + P; * H+ (ADP)

(AMP)

In thesereactions,Pi standsfor inorganic phosphate(pO+, ) and PP;for inorganic pyrophosphate,two phosphategroups linked by a phosphoanhydride bond. As the top two reactions show, the removal of a phosphate or a pyrophosphate group from ATP leaves adenosinediphosphate (ADp) or adenosinemonophosphate (AMP), respecively. A phosphoanhydride bond or other high-energy bond (commonly denoted by -) is not intrinsically different from other covalent bonds. High-energy bonds simply releaseespecially large amounts of energywhen broken by addition of water (hydrolyzed). For instance,the AG"' for hydrolysis of a phosphoanhydride bond in ATP (-7.3 kcal/mol) is more than three times the AGo' for hydrolysis of the phosphoester bond (red) in glycerol3-phosphate(-Z.2kcallmol):

ooH Ho-p-o-cHr-lH - cH2oH I

U Glycerol3-phosphate

A principal reason for this differenceis that ATp and its hydrolysis products ADP and P1are highly charged at neutral

58

CHAPTER2 I

CHEMICALFOUNDATIONS

is energeticallyfavorable (AG < 0). An alternative mechanism of energy coupling is to use the energy releasedby ATP hydrolysis to changethe conformation of the molecule to an "energy-rich" stressedstate. In turn, the energy stored as conformational stresscan be released as the molecule "relaxes" back into its unstressed conformation. If this relaxation processcan be mechanistically coupled to another reaction, the releasedenergycan be harnessedto drive important cellular processes. As with many biosynthetic reactions,transport of molecules into or out of the cell often has a positive AG and thus requires an input of energy to proceed. Such simple transport reactions do not directly involve the making or breaking of covalent bonds; thus the AGo' is 0. In the case of a substancemoving into a cell, Equation 2-7 becomes

AG: Rrt"+."9*

(2-10)

where [C1"] is the initial concentration of the substanceinside the cell and [Co",] is its concentration outside the cell. 'We can see from Equation 2-10 that AG is positive for transport of a substanceinto a cell against its concentration gradient (when [Cr"] > [C"",]); the energy to drive such "uphill" transport often is supplied by the hydrolysis of

ATP. Conversely,when a substancemoves down its concentration gradient ([C",,] > [C,"] ), AG is negative. Such "downhill" transport releasesenergythat can be coupledto an energy-requiringreaction, say, the movement of another substanceuphill acrossa membrane or the synthesisof ATP itself (seeChapters 1I and 12).

ATPls GeneratedDuring Photosynthesis and Respiration Clearlg to continue functioning, cells must constantly replenish their ATP supply. In nearly all cells,the initial energysource whose energyis ultimately transformed into the phosphoanhydride bondsof ATP and bonds in other compoundsis sunlight. In photosynthesis,plants and certain microorganismscan trap the energy in light and use it to synthesizeAIP from ADP and P1.Much of the ATP produced in photosynthesisis hydrolyzed to provide energyfor the conversionof carbon dioxide to sixcarbon sugars,a processcalledcarbon fixation: ATP ADP + P. 6co2 + 6 Hro V c 6 H 1 2 o+6 o 0 2 In animals, the free energy in sugars and other molecules derived from food is releasedin the processof respiration. All synthesisof ATP in animal cells and in nonphotosynthetic microorganismsresultsfrom the chemicaltransformation of energy-richcompounds in the diet (e.g., glucose, starch). We discussthe mechanismsof photosynthesisand cellular respiration in Chapter 12. The completeoxidation of glucoseto yield carbon dioxide, c6Flpo6 + 6 02 --->6 co2 + 6 H2o has a AG'' of -686 kcal/mol and is the reverseof photosynthetic carbon fixation. Cells employ an elaborate set of protein-mediated reactions to couple the oxidation of 1 molecule of glucoseto the synthesisof as many as 30 moleculesof ATP from 30 molecules of ADP. This oxygen-dependent (aerobic) degradation (catabolism) of glucose is the major pathway for generating ATP in all animal cells, nonphotosyntheticplant cells, and many bacterial cells. Catabolism of fatty acids can also be an important source of ATP. Light energy captured in photosynthesisis not the only source of chemical energy for all cells. Certain microorganisms that live in or around deepoceanvents,where adequate sunlight is unavailable,derive the energyfor converting ADP and Pi into ATP from the oxidation of reduced inorganic compounds.Thesereducedcompounds originate deepin the earth and are releasedat the vents.

not accompanythe formation of new chemical bonds or the releaseof energythat can be coupled to other reactions.The loss of electronsfrom an atom or a molecule is called oxidation, and the gain of electrons by an atom or a molecule is called reduction. Becauseelectrons are neither created nor destroyedin a chemical reaction, if one atom or molecule is oxidized, another must be reduced. For example' oxygen draws electronsfrom ps2+ (ferrous) ions to form Fe3* (ferric) ions, a reaction that occurs as part of the process by which carbohydrates are degraded in mitochondria. Each oxygen atom receivestwo electrons, one from each of two Fe"- ions: 2 Fe2* + Yz oz -->2 Fe3* + 02Thus Fe2*is oxidized, and 02 is reduced.Suchreactionsin which one molecule is reduced and another oxidized often are referred to as redox reactions. Oxygen is an electron acceptor in many redox reactions in cells under aerobic conditions. Many biologically important oxidation and reduction reactions involve the removal or the addition of hydrogen atoms (protons plus electrons) rather than the transfer of isolated electronson their own. The oxidation of succinate to fumarate, which also occurs in mitochondria, is an example (Figure 2-32). Protons are soluble in aqueous solutions (as H3O+), but electrons are not and must be transferred directly from one atom or molecule to another without a water-dissolvedintermediate.In this rype of oxidation reaction' electrons often are transferred to small electron-carrying molecules, sometimes referred to as coenzymes.The most common of theseelectron carriers are NAD* (nicotinamide adeninedinucleotide),which is reducedto NADH' and FAD (flavin adenine dinucleotide), which is reduced to FADH2 (Figure 2-33). The reduced forms of these coenzymescan transfer protons and electrons to other molecules, thereby reducing them.

c-oI

H-C-H | H-C-H I

c- oo

Succinate

N A D * a n d F A DC o u p l eM a n y B i o l o g i c a l Oxidation and ReductionReactions In many chemical reactions, electrons are transferred from one atom or molecule to another; this transfer may or may

o c-o

o

---\----\---2 v v ze 2H*

I c-H

c-H I

c-o o

Fumarate

to fumarate.Inthis of succinate 2-32 Conversion A FIGURE aspartof the citric in mitochondria occurs which reaction, oxidation andtwo protonsTheseare losestwo electrons acidcycle,succinate it to FADH2. to FAD,reducing transferred

B I O C H E M I C AELN E R G E T I C S

59

(b) Reduced: FADH2 Reduced: NADH

o c-NH2

+ le

I

Hll

I

Ribose I 2P

I Ribitol I 2P

Ribose I 2P

I

..1

A O e n o sn re

Adenosine

NAD++H++2e-.-

NADH

Ribitol I 2P

I

I

Adenosine

Adenosine FAD+2H- +2e-

i-

FADH2

FIGURE 2-33 Theelectron-carrying coenzymesNAD+and FAD. (a)NAD*(nicotinamide adenine dinucleotide) isreduced to NADHby the addition of two electrons andoneprotonsimultaneously In manybiological redoxreactions, a pairof hydrogen atoms(two protons andtwo electrons) areremoved froma moleculeIn some cases, oneof the protons andbothelectrons aretransferred to NAD-;the otherprotonisreleased intosolution(b)FAD(flavin

adenine dinucleotide) isreduced to FADH2 bythe additionof two electrons andtwo protons, whensuccinate asoccurs isconverted to (seeFigure fumarate 2-32)ln thistwo-stepreaction, addition of one electron together with oneprotonfirstgenerates a short-lived (notshown), semiquinone intermediate whichthenaccepts a second electron andproton.

To describeredox reactions, such as the reaction of ferrous ion (Fe2+)and oxygen (O2), it is easiestto divide them into two half-reactions:

reduce)a compound with a more positivereduction potential. In this type of reaction, the changein electricpotential AE is the sum of the reduction and oxidation potentialsfor the two half-reactions.The AE for a redox reaction is related to the changein free energyAG by the following expression:

Oxidation of Fe2n: Reduction of 02:

2 Fez* --+ 2 Fe3* -t 2 e2 e * 1/z02 -- 02-

In this case,the reducedoxygen (O2-) readily reactswith two protons to form one water molecule(HzO). The readinesswith which an atom or a molecule gains an electron is its reduction potential E. The tendencyto lose electrons,the oxidation potential, has rhe same magnitude but opposite sign as the reduction potential for the reversereaction. Reduction porentials are measuredin volts (V) from an arbitrary zero point set ar the reduction potential of the following half-reactionunder standard conditions (25 "C. 1 atm, and reactantsat 1 M):

H+ + e- IY'Z

*t,

oxidation

The value of E for a molecule or an atom under standard conditions is its standardreduction potential, E'6 A molecule or an ion with a positive E'e has a higher affinity for electronsthan the H* ion does under standardconditions. Conversely,a molecule or ion with a negative E'6 has a lower affinity for electronsthan the H* ion does under standard conditions.Like the valuesof AGo', standardreduction potentials may differ somewhat from those found under the conditions in a cell becausethe concentrations of reactants in a cell are not 1 M. In a redox reaction,electronsmove spontaneouslytoward atoms or moleculeshaving more positiuereductionporentials. In other words, a compound having a more negativereduction potential can transfer electrons spontaneouslyto (i.e.,

60

CHAPTER2 I

CHEMICALFOUNDATIONS

AG (callmol) : -n (23,064)AE (volts)

(2-11)

wheren is the number of electronstransferred.Note that a redox reactionwith a positiveAE value will have a negativeAG and thus will tend to proceedspontaneouslyfrom left to right.

BiochemicalEnergetics r The changein free energy AG is the most useful measure for predicting the direction of chemicalreactionsin biological systems.Chemical reactions tend to proceed spontaneouslyin the direction for which AG is negative.The magnitude of AG is independentof the reaction rate. r The chemicalfree-energychangeAGo' equals -2.3 RT log K.o. Thus the value of AGo' can be calculatedfrom the experimentally determinedconcentrationsof reactantsand products at equilibrium. r The rate of a reaction dependson the activation energy neededto energizereactantsto a rransition state. Catalysts such as enzymesspeedup reactions by lowering the activation energyof the transitionstate. r A chemical reaction having a positive AG can proceed if it is coupled with a reaction having a negariveAG of larger magnitude. r Many otherwise energetically unfavorable cellular processesare driven by the hydrolysis of phosphoanhydride bonds in ATP (seeFigure2-31).

r Directly or indirectlS light energycaptured by photosynthesisin plants and photosynthetic bacteria is the ultimate source of chemical energy for almost all cells. r An oxidation reaction (loss of electrons)is always coupled with a reduction reaction (gain of electrons). r Biological oxidation and reduction reactions often are coupled by electron-carryingcoenzymessuch as NADand FAD (seeFigure2-33). r Oxidation-reduction reactionswith a positive AE have a negativeAG and thus tend to proceed spontaneously.

KeyTerms acid 52

hydrophobic 3 1

a carbon atom (C*) 41

hydrophobic effect 38

amino acids41

ionic interactions 36

amphipathic 31

molecular complementarity 39

base52 buffers 52 chemicalpotential energy54

monosaccharides44

covalentbond 32

nucleotides44

dehydration r eaction 4 0 AG (free-energy change)55

oxidation 59 pH 51

disulfide bond 43

p h o s p h o a n h y d r i dbeo n d s5 7

endergonic55

phospholipid btlayers41

endothermic 55

polar 34

energy coupling 58 enthalpy (H) 55

polymer 40 redox reaction 59

entropy (S)55

reduction 59

equilibrium constant49

saturated 47

exergonic 55

steadystate50

exothermic56

stereoisomers 33

fatty acids 47

unsaturated 47

hydrogen bond 37

van der $0aals interactions37

hydrophilic 31

44 nucleosides

Review the Concepts 1,. The gecko is a reptile with an amazing ability to climb smooth surfaces,including glass.Recentdiscoveriesindicate that geckosstick to smooth surfacesvia van der'lfaals interactions between septaeon their feet and the smooth surface. How is this method of stickinessadvantageousover covalent interactions?Given that van der Sfaalsforces are among the weakest molecular interactions, how can the gecko's feet stick so effectively? 2. The K* channel is an example of a transmembraneprotein (a protein that spans the phospholipid bilayer of the plasmamembrane).What typesof amino acidsare likely to be found (a) lining the channel through which K* passes,(b) in contact with the hydrophobic core of the phospholipid bilayer

containing fatty acylgroups, (c) in the cytosolic domain of the protein, and (d) in the extracellulardomain of the protein? 3. V-M-Y-F-E-N: This is the single-letteramino acid abbreviation for a peptide. What is the net charge of this peptide can at pH 7.0? An enzyme called a protein tyrosine kinase 'Sfhat attach phosphatesto the hydroxyl groups of tyrosine. is the net charge of the peptide at pH 7.0 after it has been phosphorylated by a tyrosine kinase? \What is the likely source of phosphateutilized by the kinase for this reaction? 4. Disulfide bonds help to stabilize the three-dimensional structure of proteins. What amino acids are involved in the formation of disulfide bonds?Does the formation of a disulfide bond increaseor decreaseentropy (AS)? 5. In the 1960s, the drug thalidomide was prescribed to pregnant women to treat morning sickness. However' thalidomide caused severe limb defects in the children of some women who took the drug, and its use for morning sicknesswas discontinued.It is now known that thalidomide was administered as a mixture of two stereoisomericcomthe pounds, one of which relieved morning sicknessand 'Sfhat defects' birth for the other of which was responsible Why might two such closely related comare stereoisomers? pounds have such different physiologic effects? 6. Name the compound shown below.

HrrriS)c-)x ll :,

se C i H

;; ll ll

Hrl',t-c\fi)c-i/ o -o- P-o-o-P-o-o-?-o-9Hz I o-

I

o-

I o

5',

-o 2',

OH OH Is this nucleotide a component of DNA, RNA, or both? Name one other function of this compound. 7. The chemical basis of blood-group specificity residesin the carbohydratesdisplayedon the surfaceof red blood cells. Carbohydrateshave the potential for great structural diversity. Indeed,the structural complexity of the oligosaccharides that can be formed from four sugarsis greater than that for oligopeptidesfrom four amino acids. Sfhat propertiesof carbohydratesmake this great structural diversity possible? 8. Ammonia (NH:) is a weak basethat under acidic conditions becomesprotonated to the ammonium ion in the following reaction: NH, + H- -+ NH+NH3 freely permeates biological membranes, including those of lysosomes.The lysosomeis a subcellular organelle with a pH of about 4.5-5.0; the pH of cytoplasmis -7'0. lWhat is the effect on the pH of the fluid content of lysosomes R E V I E WT H E C O N C E P T S

61

when cells are exposedto ammonia? No/e: Protonated ammonia does not diffuse freely acrossmembranes. 9. Consider the binding reaction L + R -+ LR, where L is 'S7hen a ligand and R is its receptor. 1 x 10 3 M L is added to a solution containing5 x 10-2 M R, 90% of the L binds to form LR. rVhat is the K.o of this reaction? How will the K.o be affected by the addition of a protein that catalyzes this binding reaction?V/hat is the K6? 10. Vhat is the ionization state of phosphoric acid in the cytoplasm? Vhy is phosphoric acid such a physiologically important compound? 1 1 . T h e A G o ' f o r t h e r e a c t i o nX + Y - + X Y i s - 1 0 0 0 callmol. What is the AG at 25 'C (298 Kelvin) starting with 0.01 M each X, I and XY? Suggesttwo ways one could make this reaction energeticallyfavorable. 12. According to health experts,saturatedfatty acids,which come from animal fats, are a major factor contributing to coronary heart disease.What distinguishesa saturated fatty acid from an unsaturated fatty acid, and to what does the term saturated refer?RecentlS trans unsaturatedfatty acids, or trans fats, which raise total cholesterollevelsin the bodv. have also been implicated in heart disease.How does the cis stereoisomerdiffer from the trans configuration, and what effect does the cis configuration have on the structure of the fatty acid chain? 13. Chemicalmodifications to amino acidscontribute to the diversity and function of proteins. For instance,7-carboxylation of specific amino acids is required to make some proteins biologically active. What particular amino acid undergoes this modification, and what is the biological relevance?Warfarin, a derivative of coumarin, which is present in many plants, inhibits 7-carboxylation of this amino acid and was used in the past as a rat poison. At present, it is also used clinically in humans. What patients might be prescribedwarfarin and whv?

62

CHAPTER2 I

CHEMICALFOUNDATIONS

References Albertg R. A., and R. J. Silbey.2005. PhysicalChemistry, 4th ed. Wiley. Atkins, P.,and J. de Paula. 2005. The Elementsof Physical Chemistry,4th ed. \7. H. Freemanand Company. Berg,J. M., J. L. Tymoczko, and L. Stryer.2007. Biochemistry, 5th ed. W. H. Freemanand Company. Cantor, P. R., and C. R. Schimmel.1980. BiophysicalChemistry.V. H. Freemanand Company. Davenport,H.W. 1974. ABC of Acid-BaseChemistry,6rh ed.. University of ChicagoPress. Eisenberg,D., and D. Crothers.1,979.PhysicalChemistryuith Applications to the Life Sciences.Benjamin-Cummings. Guyton, A. C., and J. E. Hall. 2000. Textbook of Medical Physiology, 10th ed. Saunders. Hill, T. J. 1977. FreeEnergy Transductionin Biology. Academic Press. Klotz, I. M. 1978. Energy Changesin BiochemicalReactions. AcademicPress. Murray, R. K., et al. 1999.Harper'sBiocbemistry,25thed. Lange. Nicholls, D. G., and S.J. Ferguson.1992. Bioenergetics2. Academic Press. Oxtoby, D., H. Gillis, and N. Nachtrieb. 2003. Principlesof Modern Chemistry,Sth ed. Saunders. Sharon,N. 1980. Carbohydrates. Sci.Am.243(5):90-116. Tanford, C. 1980. The Hydropl;obic Effect: Formation of Micellesand Biological Membranes,2d ed. \filey. Tinoco, I., K. Sauer,and J. Wang. 200L. PhysicalChemistryPrinciplesand Applications in Biological Sciences,4thed. Prentice Hall. Van Holde, K., W. Johnson,and P.Ho. 1998. Principlesof PhysicalBiochemistry.PrenticeHall. Voet, D., and J. Voet. 2004. Biochemistry,3d,ed. \7iley. Wood, !(. B., et al. 1981,.Biochemistry:A ProblemsApproach, 2d ed. Benjamin-Cummings.

CHAPTER

STRUCTURE PROTEIN AND FUNCTION

Ribbondiagramof a betapropeller domainfrom the humansignaling (spheres) proteinKeaplTenwatermolecules areboundto eachof lvlanyproteinsarebuiltfrom multiple, the sixbladesof the propeller. X Li,C A independently stableproteindomains[FromL J Beamet Bottoms, andM Hannink,2005,ActaCrystallogr D: Biol.Crystallogr of Robert Hube[Martinsried 51(10):1335-1342I Credit.Courtesy

roteins, which are polymers of amino acids, come in many sizesand shapes.Their three-dimensionaldiversity reflects underlying structural differences: principally variations in their lengths and amino acid sequences,and in some cases,differencesalso in the number of disulfide bonds or the attachment of small moleculesor ions to their amino acid side chains. In general,the linear, unbranched polymer of amino acidscomposing any protein will fold into only one or a few closely related three-dimensional shapes-called conformations. The conformation of a protein together with the distinctive chemical properties of its amino acid side chains determines its function. As a consequence,proteins can perform a dazzling array of distinct functions inside and outside of cells that either are essentialfor life or provide selective evolutionary advantageto the cell or organism that contains them. It is, therefore, not surprising that characterizing the structuresand activities of proteins is a fundamental prerequisitefor understanding how cells work. Much of this textbook is devoted to examining how proteins act together to enablecells to live and function properly. Many proteins can be grouped into just a few broad func. Structural proteins, for example,determinethe tional classes shapesof cellsand their extracellularenvironments,and serve as guide wires or rails to direct the intracellularmovement of moleculesand organelles.They usually are formed by the assemblyof multiple protein subunitsinto very large,long strucnres. Scaffold proteins bring other proteins together into

ordered arrays to perform specific functions more efficiently than if those proteins were not assembledtogether. Enzymes are proteins that catalyze chemical reactions. Membrane transport proteins permit the flow of ions and molecules acrosscellular membranes. Regulatoryproteins act as siSnals' sensors,and switches to control the activities of cells by altering the functions of other proteins and genes.These include signaling proteins, such as hormones and cell-surfacereceptors that transmit extracellular signals to the cell interior'

OUTLIN E 3.1

HierarchicalStructureof Proteins

64

3.2

P r o t e i nF o l d i n g

74

3.3

ProteinFunction

78

3.4

RegulatingProteinFunction ProteinDegradation

3.5

R e g u l a t i n gP r o t e i nF u n c t i o nl l : Noncovalentand CovalentModifications

3.6

Purifying,Detecting,and CharacterizingProteins

3.7

Proteomics

92 105

63

Motor proteins are responsiblefor moving other proteins, organelles,cells-even whole organisms.Any one protein can be a member of more than one protein class,as is the caseof some cell-surfacesignaling receptorsthat are both enzymes and regulator proteins becausethey transmit signals from outside to inside cells by catalyztngchemical reactions. To accomplishefficientlytheir diversemissionssomeproreinsassembleinto largecomplexes,often calledmolecularmachines. How do proteinsmediateso many diversefunctions?They do this by exploiting a few simple activiries.Most fundamentally, proteins bind-to one anorher,to other macromolecules, suchas DNA, and to smallmoleculesand ions.In many cases such binding can induce a conformational changein the protein and thus influenceits activity. Binding is basedon molecular complementaritybetweena protein and its binding partner, as describedin Chapter 2. A second key activity is enzymatic catalysis.Appropriate folding of a protein will place some amino acid side chainsand carboxyl and amino groups of the backboneinto positions that permit the catalysis of covalent bond rearrangements.A third activity involves folding into a channel or pore within a membrane through which moleculesand ions flow. Although theseare especially crucial protein activities,they are not the only ones. For example,fish that live in frigid waters-rhe Antarctic borchsand Arctic cods-have antifreezeproteins in their circulatory systems to preventwater crystallizationat subzerotemperatures. A completeunderstandingof how proteins permit cells to live and thrive requiresthe identificationand characterization of all the proteins used by a cell. In a sense,molecular cell biologistswant to compile a completeprotein 'parts list' and consrructan all-inclusive"users manual" that describeshow theseprclteinswork. Compiling a comprehensive protein parts list has become feasiblein recent years with the sequencingof entire genomes-complete sets of g e n e s - o f m o r e a n d m o r e o r g a n i s m s .F r o m a c o m p u r e r analysisof genome sequences,researcherscan deducethe number of amino acidsand their sequenceof most of the encoded proteins (Chapter5). The rerrn proreomewas coined to refer to the entire protein complementof an organism.The human genome conrains 20,000-25,000 genes(only four times that of the single-cellyeast Saccharomycescereuisiae). However, it encodesabout 33.000 different protein because of variation in mRNA producrion (e.g..alteinativesplicing (Chapter8)). Even more variation is generaredby 100 types of protein modification that can produce hundreds of thousandsof distinct human proteins.By comparingprotein sequencesand structures of proteins of unknown function to those of known function, scientistscan often deduce much about their functions. In the past, characterizationof protein function by genetic, biochemical, or physiological methods often precededthe identification of particular proteins. In the moderngenomicand proteomicera,a protein is usuallyidentified prior to determining its function. In this chapter,we begin our study of how the structureof a protein givesriseto its function, a themethat recursthroughout this book (Figure 3-1). The first secrion examineshow chains of amino acid building blocks are arrangedin a threedimensional structural hierarchy.The next section discusses 64

CHAPTER 3

|

PROTEIN S T R U C T U RAEN D F U N C T I O t T

MOLECULAR STRUCTURE Primary (sequence)

S e c o n d a r y( l o c a lf o l d i n g )

Tertiary ( l o n g - r a n gf e olding)

Supramolecular (large-scale assembly)

Ouaternary (multimericstructure)

(b)

Regulation

@ FUNCTION Transport i

C a t a l y s i s4

FIGURE 3-1 Overviewof proteinstructureand function. (a)Proteins areassembled according to a hierarchy of structures A polypeptide l i 'nse asr e q u e n coef a m i n oa c i d sl i n k e db y p e p t i d e bonds(primary structure) foldsinto localhelices or sheets (secondary structure) thatpackrntolarge(longer-range) complex (tertiary three-dimensional structures structu re) Someindividual polypeptides (quaternary associate intomultichain complexes structure), whichin somecases canbeverylarge,consisting of tensto (supramolecular (b)Protein hundreds of subunits assemblies) function includes organization of thegenome, otherproteins, lipidbilayer (structure); membranes, andcytoplasm controlof proteinactivity (regulation), monitoring of theenvironment andtransmitting resultant (signaling), information flowof smallmolecules andionsacross (transport); membranes (viaenzymes); catalysis of chemical reactions (viamotorproteins) andgeneration of forcefor movement These functions andothersarisefromspecifrc bindinginteractions and conformatronal changes in thestructure of a properly foldedprotein how proteins fold into thesestructures.\(e then rurn to protein function, focusingon enzymes,the specialclassof proteinsthat catalyzechemicalreactions.Variousmechanismsthat cellsuse to control the activitiesand life spansof proteinsare coveredin the next two sections.Next comesa sectionon commonly used techniquesin the biologist'stool kit for isolating proteins and characterizingtheir properties.The chapter concludeswith a discussionof the burgeoningfield of proteomics.

ff,t

Hierarchical Structureof Proteins

A protein chain folds into a distinct three-dimensionalshape that is stabilizedby noncovalent interacrionsberween regions in the linear sequenceof amino acids.A kcy concept rn

(a) Primarystructure -Ala -Glu -Val-Thr-Asp- Pro-Gly-

(b) Secondarystructure s heli

(c) Tertiarystructure

Domain

bond formation between the amino group of one amino acid and the carboxyl group of another results in the net releaseof a water molecule (dehydration) (Figure 3-3a). The repeated amide N, cr carbon (C*), carbonyl C and oxygen atoms of each amino acid residue form the backbone of a protein molecule from which the various side-chaingroups project (Figure 3-3b, c). As a consequenceof the peptide linkage, the backbone exhibits directionality becauseall the amino groups are located on the same side of the Co atoms. Thus one end of a protein has a free (unlinked) amino group (the N-terminus), and the other end has a free carboxyl group (the C-terminus). The sequenceof a protein chain is conventionally written with its

(d) Ouaternarystructure (a)

HO tll

HO

ttl

*HsN- Cd-CR1

O- + *H3N- Co-C-

3l

ll lN t,o \7

O-

n'

HOHO

riltll

(a)Thelinear FIGURE 3-2 Fourlevelsof proteinhierarchy. peptide bondsisthe acids linked together by of amino sequence (b)Folding primary chainintolocala structure. of the polypeptide (c)Secondary represents secondary structure. helices or B sheets loopsandturnsin a single together with various structural elements polypeptide stablestructure, chainpackintoa largerindependently distinct domains; thisistertiarystructure(d) whichmayinclude polypeptides can with theirown tertiary structures Someindividual complex. a multichain intoa quaternary structure defining associate understandinghow proteins work is that function is deriued from three-dimensional structure, and three-dimensional structure, which is determined primarily by noncoualent interactions betueen regions in the linear sequenceof amino acids, is specifiedby amino acid seqwence.Indeed,principles relating biologicalstructureand function initially were formulated by the biologistsJohannvon Goethe(1'749-1,832),Ernst Haeckel (1834-1.91.9),and D'Arcy Thompson \1'860-1'948). They greatly influencedthe school of "organic" architecture pioneeredin the early rwentieth century that is epitomizedby the dicta "form follows function" (Louis Sullivan)and "form is function" (Frank Lloyd'Wright). Here, we considerthe architectureof proteins at four levelsof organization: primar5 secondary,tertiary, and quaternary (Figure 3-2).

The PrimaryStructureof a Proteinls lts Linear Arrangemeno t f AminoAcids As discussedin Chapter 2, proteins are constructed by the polymerizationof 20 differenttypesof amino acids.Individual amino acids are linked together in linear, unbranched chains by covalent amide bonds, called peptide bonds, with occasional disulfide bonds covalently linking side chains together.Peptide

PePtide bond (b)

(C-terminus)

(N-terminus) (c)

amino 3-3 Structureof a polypeptide.(a)Individual A FIGURE via reactions form which peptide bonds, by together linked are acids the R1,R2,etc.,represent thatresultin a lossof water(dehydration). polymers of ("Rgroups")of aminoacids.(b)Linear sidechains whichhave aminoacidsarecalledpolypeptides, oeptidebond-linked (C-terminus) (N-terminus) end carboxyl free and a a freeaminoend linkingthe (c)A ball-and-stick modelshowspeptidebonds(yellow) atom(blue)of oneaminoacid(aa)with thecarbonyl aminonitrogen onein thechain.TheR groups of an adjacent carbonatom(gray) (black) of theaminoacids atoms (green) cr carbon extendfromthe properties of distinct the determine largely chains side These individual oroteins EF P R O T E I N S H I E R A R C H I C ASLT R U C T U RO

65

N-terminal amino acid on the left and its C-terminal amino acid on the right, and the amino acids are numbered sequentially starting from the amino terminus (number 1). The primary structure of a protein is simply the linear arrangement, or sequence,of the amino acid residuesthat compose it. Many terms are used to denote the chains formed by the polymerization of amino acids. A short chain of amino acids linked by peptide bonds and having a defined sequenceis called an oligopeptide, or just peptide; longer chains are referred to as polypeptides. peptides generally contain fewer than 20-30 amino acid residues, whereas polypeptides are often 200-500 residueslong. The longest protein described to date is the muscle protein titin with 25,926 residues.We generally reservethe term protein for a polypeptide (or complex of polypeptides) that has a welldefined three-dimensionalstructure. It is implied that proteins and peptides are the natural products of a cell. The size of a protein or a polypeptide is reported as its massin daltons (a dalton is 1 atomic massunit) or as its molecular weight (M\7), which is a dimensionlessnumber. For example,a 10,000-MI7 protein has a mass of 10,000 daltons (Da), or 10 kilodaltons (kDa). In the penultimatesection of this chapter, we will consider different methods for measuringthe sizesand other physical characteristicsof proteins. The known and predicted proteins encoded by the yeast genome have an averagemolecular weight of 52,728 and contain, on average,466 amino acid residues.The average molecular weight of amino acids in proteins is 113, taking into account their averagerelative abundances.This value can be used to estimate the number of residuesin a protein from its molecular weight or, conversely,its molecular weight from the number of residues.

SecondaryStructuresAre the Core Elements of ProteinArchitecture The secondlevel in the hierarchy of protein strucrure is secondary structure. Secondary structures are stable spatial arrangementsof segmentsof a polypeptide chain held together by hydrogen bonds between backbone amide and carbonyl groups and often involving repeatingstructural patterns. A singlepolypeptide may contain multiple types of secondary structure in various portions of the chain, depending on its sequence.The principal secondarystructures are the alpha (c) helix, the beta (p) sheet,and a short U-shapedbeta (F) turn. Portions of the polypeptide that don't form these structures,but neverthelesshave a well-defined,stableshape, are said to have anirregular structure.The term random ioil appliesto highly flexible portions of a polypeptide chain that have no fixed three-dimensionalstructure.In an averageprotein, 50 percent of the polypeptide chain exists as ct helices and B sheets;the remainder of the molecule is in coils and turns. Thus, ct helicesand B sheetsare the major internal supportive elementsin most proteins. In this section,we exploie the shapesof secondarystructuresand the forces that iavor their formation. In later sections,we examine how linear arrays of secondary structure fold together into larger, more complex arrangementscalled tertiary structure. 66

.

c H A p r E 3R |

pRorEtN s r R u c r u RA EN DF U N c l o N

A m i n ot e r m i n u s

Carboxylterminus

A FIGURE 3-4 The ct helix, a commonsecondarystructurein proteins.Thepolypeptide (seenasa ribbon)isfoldedinto backbone a spiralthat isheldin placeby hydrogen bondsbetweenbackbone oxygenandhydrogen atomsOnlyhydrogens involved in bondingare shown.Theoutersurface of the helixiscovered bv theside-chain R groups(green)

The a Helix In a polypeptide segmentfolded into an o helix, the backbone forms a spiral structure in which the carbonyl oxygen atom of eachpeptide bond is hydrogen-bonded to the amide hydrogen atom of the amino acid four residues farther along the chain (in the direction of the C-terminus) (Figure 3-4). Within an a helix, all the backbone amino and carboxyl groups are hydrogen-bondedto one another,exceptat the very beginning and end of the helix. This periodic arrangementof bonds confersan amino-to-carboxy-terminal directionality on the helix becauseall the hydrogen bond acceptors (e.g.,the carbonyl groups) have the same orientation (pointing in the downward direction in Figure 3-4) and results in a structure in which there is a complete turn of the spiral every 3.6 residues.An ct helix 36 amino acids long has 10 turns of the helix and is 5.4 nm long (0.54 nm/turn). The stable arrangement of hydrogen-bonded amino acids in the a helix holds the bar:kbone in a straight, rodlike cylinder from which the side chains point outward. The relative hydrophobic or hydrophilic quality of a particular helix within a protein is determined entirely by the

characteristicsof the side chains, becauseall the polar amino and carboxyl groups of the peptide backboneare engagedin hydrogen bonding with one another in the helix. In watersoluble proteins, the hydrophilic helicestend to be found on the outside surfaces,where they can interact with the aqueous environment, whereas hydrophobic helices tend to be buried within the core of the folded protein. The amino acid proline is usually not found in cr helices,becausethe covalent bonding of its amino group with a carbon in the side chain preventsits participation in stabilizingthe backbonethrough normal hydrogen bonding. While the classic ct helix is the most intrinsically stable, and most common helical form in proteins, there are variations, such as more tightly or loosely twisted helices.For example, in a specializedhelix called a coiled coil (describedseveralsectionsfarther on), the helix is more tightly wound (3.5 residuesand 0.51 nm per turn). The p Sheet Another type of secondarystructure, the B sheet,consistsof laterally packed B strands.Each B strand is a short (5- to 8-residue),nearly fully extended polypeptide segment. Unlike in the ct helix (where hydrogen bonding between the amino and carboxyl groups in the backbone occursbetlveen nearly adjacentresidues),hydrogenbonding in the B sheetoccurs between backbone atoms in separate,but adjacent, B strands (Figure 3-5a). Thesedistinct B strandsmay be either within a single polypeptide chain, with short or long loops be-

(a) Topview

'lo e

"{r ...)

Amino termrnus

Carboxyl termrnus

(b) Side

A FIGURE 3-5 The p sheet,anothercommonsecondary structurein proteins.(a)Topviewof a simplethree-stranded B p strandsThestabilizing rrydrogen bonds sheetwith antiparallel lines(b)Side by greendashed areindicated the B strands between (green) projection groups aboveand R of the viewof a B sheetThe in thisview Thefixedbond belowthe planeof thesheetisobvious produce contour a pleated backbone anglesin the polypeptide

of four residues, 3-6 Structureof a p turn' Composed A FIGURE chain(=180"U-turn)The of a polypeptide thedirection B turnsreverse <0.7 nmapart, areusually of thefirstandfourthresidues C. carbons bond. a hydrogen areoftenlinkedby B turns andthoseresidues structures polypeptides into compact of long folding the facilitate

Nveenthe B strand segments'or on different polypeptide chains. Figure 3-5b shows how two or more B strands align into adiacent rows, forming a nearly two-dimensional B pleatedsheet(or simply pleated sheet), in which hydrogen bonds within the plane of the sheethold the B strandstogether as the side chains itick out aboveand below the plane. Like a helices,B strands have a directionality defined by the orientation of the peptide bond. Therefore, in a pleated sheet,adjacent p strands can be oriented in the same (parallel) or opposite (antiparallel) directions with respectto eachother' In someproteins, B sheetsform the floor of a binding pocket or a hydrophobic core; in other oroteins embedded in membranes the B sheetscurve around and form a hydrophilic central pore through which ions and small moleculesmay flow (Chapter11). p Turns Composedof four residues,B turns are locatedon the surface of a protein, forming sharp bends that reversethe direction of the polypeptide backbone, often toward the protein's interior. These short, U-shaped secondarystructures are often stabilizedby a hydrogenbond betweentheir end residues (Figure 3-6). Glycine and proline are commonly present in turns. The lack of alargeside chain in glycine and the presence of a built-in bend in proline allow the polypeptidebackboneto fold into a tight U shape.B turns help large proteins to fold into highly compact structures.There are six types of well-defined turns, their detailed structures depending on the arrangement of H-bonding interactions.A polypeptide backbonealso may contain longer bends, or loops' In contrast with tight B turns, which exhibit just a few well-defined conformations' longer loops can have many different conformations.

O v e r a l lF o l d i n go f a P o l y p e p t i d eC h a i nY i e l d sl t s TertiaryStructure Tertiary structure refers to the overall conformation of a polypeptide chain-that is, the three-dimensionalarrange-..ri oi all its amino acid residues'In contrastwith secondary EF P R O T E I N S H I E R A R C H I C ASLT R U C T U RO

67

Animation:Oil Drop Model of ProteinStructurefntt

Unfoldedprotein

Nativeprotein

FIGURE 3-7 Oil drop modelof proteinfolding.The hydrophobic residues of a polypeptide chaintendto cluster together, somewhat likean oil drop,on the inside, or core,of a foldedprotein, drivenawayfromthe aqueous surroundings bythe hydrophobic effect(Chapter 2) Charged polarside anduncharged chains appearon the protein's surface wheretheycanform stabilizing interactions with surroundinq waterandions structures, which are stabilized only by hydrogen bonds, tertrary structure is primarily stabilized by hydrophobic interactions between nonpolar side chains, together with hydrogen bonds between polar side chains and peptide bonds. These stabilizing forces compactly hold together elements of secondary structure-o helices, B strands, turns, and coils. Becausethe stabilizing interactions are weak, however, the tertiary structure of a protein is not rigidly fixed but undergoescontinual, minute fluctuations, and some segmentswithin the tertiary structure of a protein can be so very mobile they are consideredto be disordered (that is, lacking well-defined, stable,three-dimensionalstructure). This variation in structure has important consequencesfor the function and regulation of proteins. _ Chemical properties of amino acid side chains help define tertiary structure. Disulfide bonds between the side chains of cysteineresiduesin some proteins covalently link regions of proteins, thus restricting the mobility of proteins and increasingthe stability of their tertiary structures.Amino acids with charged hydrophilic polar side chains tend to be on the outer surfacesof proteins; by interacting with water, they help to make proteins soluble in aqueoussolutions and can form noncovalent interactions with other water-soluble molecules,including other proteins. In contrast, amino acids with hydrophobic nonpolar side chains are usually sequesteredaway from the water-facing surfacesof a protein, in many casesforming a water-insolublecentral core (called the oil drop model of globular proteins, becauseof the relatively hydrophobic, or 'oily', core, Figure 3-7). Uncharged hydrophilic polar side chains are found on both the surface and inner core of proteins.

do not readily dissolvein water, usually play a structural role or participate in cellular movements.Globwlar proteins are generally water-soluble, compactly folded structures, often but not exclusively spheroidal, that comprise a mixture of secondary structures (seethe structure of myoglobin, below). Integral membrane proteins are embedded within the phospholipid bilayer of the membranes that serve as the walls of cells and organelles.The three broad categoriesof proteins noted here are not mutually exclusive-some proteins are made up of combinations of two or even all three of thesecategories.

Different Ways of Depictingthe Conformation of ProteinsConveyDifferent Typesof Information The simplest way to representthree-dimensionalprotein structure is to trace the courseof the backbone atoms, sometimes only the C. atoms, with a solid line (called a Co trace, Figure 3-8a); the most complex model shows every atom (Figure 3-8b). The former shows the overall fold of the polypeptide chain without consideration of the amino acid side chains; the latter, a ball-and-stick model, details the interactions between side-chain atoms, including those that stabilize the protein's conformation and interact with other molecules, as well as the atoms of the backbone. Even though both views are useful, the elements of secondary structure are not always easily discernedin them. Another type of representationusescommon shorthand symbols for depicting secondarystructure-for example, coiled ribbons or solid cylinders for o' helices,flat ribbons or arrows for B strands,and flexible thin strandsfor B turns, coils, and loops (Figure3-8c). In variations of ribbon diagrams,ball-and-stick or space-fillingmodels of side chains can be attached to the backbone ribbon, while ribbon and cylinder models make the secondarystructuresof a protein easyto see. However, none of these three ways of representingprotein structure conveys much information about the protein surface,which is of interest becauseit is where other molecules usually bind to a protein. Computer analysiscan identify the surface atoms that are in contact with the watery environment. On this water-accessible surface,regionshaving a common chemical character (hydrophobicity or hydrophilicity) and electricalcharacter (basic or acidic) can be indicated by coloring (Figure 3-8d). Such models reveal the topography of the protein surface and the distribution of charge, both important features of binding sites, as well as clefts in the surface where small molecules bind. This view representsa protein as it is "seen" by another molecule.

S t r u c t u r aM l o t i f s A r e R e g u l a rC o m b i n a t i o n o sf Secondaryand TertiaryStructures Particular combinations of secondary and tertiary structures, called structural motifs or folds, appear often as segments within many different proteins. Structural motifs 58

o

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pRorEtN s r R u c r u RA EN DF U N c l o N

(a) Co backbonetrace

(b) Ball and stick

( c )R i b b o n s

(d) Solvent-accessible surface

contribute to the global structure of the entire protein' and any particular structural motif often performs a common function in different proteins (e.g., binding to a particular small molecule or ion). The primary sequencesresponsible for any given structural motif may be very similar to one another. In other words, a common sequencemotif can result in a common three-dimensionalstructural motif. However, it is possible for seemingly unrelated primary sequencesto result in folding into a common structural motif. Conversely, it is possiblethat a commonly occurring sequencemotif does not fold into a well-defined structural motif. Sometimes short sequencemotifs that have an unusual abundanceof a particular amino acid, e.g., proline or aspartateor glutamate, are called "domains"l however,these and other short contiguous segmentsare more appropriately called motifs than domains (which are defined below). Many proteins, including fibrous proteins and DNAregulating proteins called transcription factors (Chapter 7), assembleinto dimers or trimers by using an ct helix-based coiled coil, or heptad-repeat,structural motif. In this structural motif, ct helicesfrom two, three, or even four separate polypeptide chains coil about one another-resulting in a coil of coils, hence the name (Figure 3-9a). The individual helicesbind tightly to one another becauseeach helix has a strip of aliphatic (hydrophobic, but not aromatic) side chains (leucinevaline, etc.) running along one side of the helix that interacts with a similar strip in the adjacent helix, thus sequesteringthe hydrophobic groups away from water

3-8 Fourways to visualizeprotein < FIGURE guanine Ras, structure. a monomertc protein, isshownin allfour nucleotide-binding (GDP) always panels, diphosphate with guanosine (a) trace in blue TheC* backbone depicted istightly howthe polypeptide demonstrates (b)A ball-and-stick packedintoa smallvolume. of allatoms. the location reveals representation how B (c)A ribbonrepresentation emphasizes (red)are (lightblue)ando helices strands in the proternNotetheturnsand organized (d) pairsof helices andstrands. loopsconnecting surfacereveals A modelof the water-accessible on the lumps,bumps,andcrevices the numerous positive are charge of proteinsurfaceRegions chargeare of neqative purple;regions shaded red shaded

and stabilizing the assemblyof multiple independenthelices' These hydrophobic strips are generatedalong only one side of the helix becausethe primary sequencesof the helicesexhibit a motif of repeating segmentsof seven amino acids (heptads) in which the sides chains of the first and fourth residuesare aliphatic and the other side chains are often hydrophilic (Figure 3-9a). Becausehydrophilic side chains ext..,J fro- one side of the helix and hydrophobic side chains extend from the opposite side,the overall helical structure is amphipathic. Becauseleucine frequently appearsin the fourth poritionr and the hydrophobic side chains merge together iike the teeth of a zipper, these structural motifs are also called leucine zippers. Many other structural motifs employ a helices' A common calcium-binding motif called the EF hand uses two short helicesconnectedby a loop (Figure 3-9b). This structural motif found in more than 100 proteins is used for sensing the calcium levels in cells. The bindittg of a Ca2* ion to oxygen atoms in conservedresiduesin the loop dependson the concentration of Caz* and often induces a conformational change in the protein, altering its activity. Thus, calcium concentrationscan directly control proteins' structures and functions. Somewhat different helix-turn-helix and basic helixloop-helix (bHLH) structural motifs are used for protein binding to DNA and consequentlythe regulation of geneactivity. Yet another motif commonly found in proteins ihat bind RNA or DNA is the zinc finger, which contains three secondary structures-an ct helix and two B strands EF P R O T E I N S . H I E R A R C H I C ASLT R U C T U RO

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(a) Coiled-coilmotif

(b) EF-hand/helix-loop-helix motif

(c) Zinc-fingermotif

Ca2*

Leu (4) Asn(1)

FIGURE 3-9 Motifs of protein secondarystructure.(a)The parallel two-stranded coiled-coil motif(left)ischaracterized by two o helices woundaroundeachother.Helixpacking isstabilized by interactions betweenhydrophobic (redandblue)present sidechains at regular intervals alongeachstrand,andfoundalongtheseamof the intertwined helices. Eacho helixexhibits a characteristic heotad repeatsequence with a hydrophobic residue often,but not always, at positions 1 and4, asindicated Thecoiled-coil natureof this structural motifismoreapparent in longcoiledcoils(Rrghf drawnat different scale)(b)An EFhanda typeof helix-loop-helix motif, consists of two helices connected by a shortloopin a specific conformation commonto manyproteins, including manycalciumbindingandDNA-binding regulatory proteinsIn calcium-bindinq

with an antiparallel orienrarion-that form a fingerlike bundle held together by a zinc ion (Figure 3-9c). rWewill encounter numerous additional modfs in later discussionsof other proteins in this and other chapters.The presenceof the same structural motif in different proteins with similar functions clearly indicates that these useful combinations of secondary structures have been conserved in evolution.

S t r u c t u r aal n d F u n c t i o n aD l o m a i n sA r e M o d u l e s of TertiaryStructure Distinct regions of protein tertiary structure are often referred

70

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proteins suchascalmodulin, oxygenatomsfromfiveresidues in the acidicAlutamateandaspartate-rich loopandonewatermolecule formionrcbondswith a Ca2*ion.(c)Thezinc-finger motifispresent in manyDNA-binding proteins that helpregulate transcription. A 7n'- ionis heldbetween (blue)anda singleo a pairof B strands helix(red)by a pairof cysteine residues anda pairof histidine residues Thetwo invariant cysteine residues areusually at positions 3 and6, andthetwo invariant histidine residues areat positions 20 and24 in this25-residue motif.[See A. Lewit-Bentley andS Rety, 2000, EF-hand calcium-binding proteins, Curr. OpinStrucBiol.10:637-643; SA W o l f e , L N e k l u d o v aa, n d C O P a b o ,2 0 0 0 , D N A r e c o g n i t i o nb y C y s 2 H r sz2i n c finger proteins,Ann Rev.Biophys Biomol. Struc.29:1g3-2121

group to another molecule) or binding ability (e.g.,a DNAbinding domain or a membrane-bindingdomain). Functional domains are often identified experimentally by whittling down a protein to its smallestactive fragment with the aid of proteases,enzymesthat cleaveone or more peptide bonds in a target polypeptide.Alternatively, the DNA encoding a protein can be modified so that when the modified DNA is used to generatea protein, only a particular region, or domain, of the full-length protein is made. Thus it is possible to determine if specificportions of a protein are responsiblefor particular activities exhibited by the protein. Indeed, functional domains are often also associatedwith correspondingstructural domains. A structural dornain is a region =40 or more amino acids in length, arrangedin a stable,distinct secondaryor tertiary structure, that often can fold into its characteristic structure independently of the rest of the protein. As a consequence.distinct structural domains can be linked together-sometimes by short or long spacers-to form a large, multidomain protein. Each of the subunits in hemagglutinin, for example, contains

Globular domain

Fibrous domain

PROXIMAL

N External Viral membrane lnternal

3-10 Tertiaryand quaternarylevels < FIGURE of structure.Theprotetnpicturedhere, of (HA),isfoundon the surface hemagglutinin has molecule virus.Thislong,multimeric influenza of two eachcomposed subunits, threeidentical HA1andHA2(a)Tertiary polypeptide chains, thefolding HA subunitcomprises of each structure structure intoa compact andstrands of itshelices intotwo domains' thatis 13.5nm longanddivided isfoldedintoa domain(silver) Themembrane-distal membrane-proximal The globular conformation conformation stemlike domain(gold)hasa fibrous, of two longcthelices owingto thealignment in HAl Shortturns (cylinders) of HA2with B strands and longerloops,oftenat the surfaceof the in each andstrands the helices connect molecule, by of HAisstabilized structure chain.(b)Quaternary betweenthe longhelices lateralrnteractions of thethree (cylinders) in thefibrousdomains forminga triple(gold,blue,andgreen), subunits stalk.Eachof the distalglobular coiled-coil stranded in HA bindssialicacid(red)on thesurface domains proteins, HA of targetcellsLikemanymembrane carbohydrate linked covalently several contains (notshown). chains

t

,

I I

t

I I I

a globular domain and a fibrous domain (Figure 3-10a)' Like structural motifs (composed of secondary structures)' structural domains (composed of secondary and tertiary structures) are incorporated as modules into different proteins. The modular approach to protein architecture is particularly easyto recognizein large proteins, which tend to be mosaics of different domains that confer distinct activities and thus can perform different functions simultaneously. Structural domains frequently are also functional domains in that they can have an activity independentof the rest of the protein. In Chapter 6 we consider the mechanism by which the gene segmentsthat correspond to domains becameshuffled in the course of evolution, resulting in their appearance in many proteins. The epidermal growth factor (EGF) domain is a structural domain presentin severalproteins (Figure3-11). EGF is a small, soluble peptide hormone that binds to cells in the embryo and in skin and connectivetissue in adults, causing them to divide. It is generatedby proteolytic (breaking of peptide bond) cleavagebetween repeated EGF domains in the EGF precursor protein, which is anchored in the cell domain. EGF domains membraneby a membrane-spanning with sequencessimilar to, but not identical with, those in the EGF peptide hormone are present in other proteins and can be liberated by proteolysis.Theseproteins include tissue plasminogenactivator (TPA), a proteasethat is used to dissolve blood clots in heart attack victims; Neu protein, which takes part in embryonic differentiation; and Notch protein, a receptor protein in the plasma membrane that

functions in developmentally important signaling (Chapter 16). Besidesthe EGF domain, theseproteinshave other domains in common with other proteins. For example, TPA

EGF precursor

EGF

A FIGURE3-11 Modular nature of protein domains' Epidermal

andP Bork,1993'Curr' shapeand color.lndaptedfromI D Campbell Opin StrucBiol 3.3851 EF P R O T E I N S O H I E R A R C H I C ASLT R U C T U RO

71

the domains in all proteins. Structural domains can be recognized in proteins whose structures have been determined by x-ray crystallography or nuclear magnetic resonance (NMR) analysis or in images captured by electron microscopy. Regions of proteins that are defined by their distinctive spatial relationshipsro the rest of the protein aretopological domains. For examplc, some prorerns associatedwith iellsurfacemembranescan have a portion extending inward into the cytoplasm (cytoplasmic domain), a portion embedded within the phospholipid bilayer membrane (membranespanningdomain), and a portion extending outward into the extracellular space(extracellulardomain). Each of thesecan comprlse one or more structural motifs and structural and functional domains.

ProteinsAssociateinto MultimericStructures a n d M a c r o m o l e c u l aArs s e m b l i e s Multimeric proteins consisr of two or more polypeptide chains or subunits. A fourth level of structur;l orga-nization, quaternary structure, describes the number (stoi_ chiometry) and relative positions of the subunits in multimenc proteins. Hemagglutinin, for example,is a trimer of three identical subunits (homotrimer) held together by noncovalent bonds (Figure 3-10b). Other multimeric pro_ teins can be composed of various numbers of ideniical (homomeric) or different (hereromeric)subunits (see the discussionof hemoglobin, below). Often, the individual monomeric subunits of a multimeric protein cannot func_ tion normally unlessthey are assembledinto the multimeric protein. In some cases,assemblyinto a multimeric protein (oligomerization)permits proreins that actsequentiallyin a pathway to increasetheir efficiencyof operaiion owing to their juxtaposition in space. The highest level in the hierarchy of protein strucrure is the associationof proteinsinto macromolecularassemblies. Typicallg such structures are very large, in some casesexceeding1 MDa in mass,approaching30-300 nm in size,and containing tens to hundreds of polypeptide chains, and sometimesother biopolymers such as nucleic acids.The cap_ sid that encasesthe nucleic acidsof the viral genomeis an ex_ ample of a macromolecular assemblywith a'structural function. The bundles of cytoskeletalfilaments that support and give shape to the plasma membrane are another examole. Other macromolecular assembliesact as molecular machines, carrying out the most complex cellular processesbv integrating individual functions into one coordinared

tional components including general transcription factors, promoter-binding proteins, helicase,and other protein comp l e x e s( F i g u r e3 - 1 2 ) . R i b o s o m e sa. l s o d i s c u s s e d in Chapter 4, are complex multiprotein and multi-nucleic acid machines that synthesizeproteins. 72

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Generaltranscriptionfactors

R N Ap o l y m e r a s

DNA Promoter I

I

Y

Transcription preinitiation comprex

A FfGURE 3-12 A macromolecular machine:the transcriptioninitiationcomplex.ThecoreRNApolymerase, general transcription factors, a mediator complex containing about20 subunits, andother proteincomplexes not depicted hereassemble at a promoter in DNA Thepolymerase carries out transcription of DNA;theassociated proteins arerequired for initialbindingof polymerase to a specific promoterThemultiple components functiontoqetherasa machine

M e m b e r so f P r o t e i nF a m i l i e sH a v ea C o m m o n E v o l u t i o n a r yA n c e s t o r Studiesof myoglobin and hemoglobin, the oxygen-carrying proteins in muscle and red blood cells,respectively,provided early evidencethat a protein's function derivesfrom its threedimensional structure, which in turn is specified by amino acid sequence.X-ray crystallographic analysis showed that the three-dimensionalstructuresof myoglobin (a monomer) and the cr and B subunitsof hemoglobin (a o2g2retramer) are remarkably similar. Sequencingof myoglobin and the hemoglobin subunits revealedthat many identical or chemically similar residues are found in identical positions throughout the primary structures of both proteins. A mutation in the gene encoding the B chain that resultsin the substitution of a valine for a glutamic acid disturbs the fold, ing and function of hemoglobin and causes sickle-cell anemla. Similar comparisonsbetweenother proteins conclusively confirmed the relation between the amino acid sequence, three-dimensionalstructure, and function of proteins. Use of sequencecomparisons to deduce protein function has ex_ panded substantiallyin recent years as the genomesof more and more organismshave been sequenced. The molecular revolution in biology during the last decadesof the twentieth century also created a new scheme of biological classification based on similarities and differencesin the amino acid sequencesof proteins. proteins that have a common ancestor are referred to as homologs. The main evidencefor homology among proteins, and hence for their common ancestry,is similarity in their sequencesor

Monocot hemoglobin

Dicot hemoglobin

Annelid

Hemoglobin Protozoan Algal F un g aI Bacterial

Ancestral ygen-binding

Ps u b u n i t of hemoglobin

Myoglobin

3-13 Evolutionof the globin proteinfamily.leff: A FIGURE globinisthoughtto bethe primitive oxygen-binding monomeric and myoglobins, muscle hemoglobins, blood modern-day of ancestor that haverevealed comparisons Sequence plantleghemoglobins and of animals parallels the evolution of the globinproteins evolution of plantglobins with thedivergence occurred plantsMajorjunctions Latergene fromhemoglobin andof myoglobin fromanimalglobins

Rrght: of hemoglobin gaveriseto theo andB subunits duplication of two a andtwo B subunitsThe isa tetramer Hemoglobin and with leghemoglobin of thesesubunits similarity structural A heme is evident monomers, are which of both myoglobin, with eachglobinpolypeptide (red)noncovalently associated molecule in theseproteins[(relt) for oxygen-binding responsible isdirectly 93:5675 l AcadSciIJSA Nat'l Proc 1996, fromR C Hardison, Adapted

'We can therefore describehomologous proteins structures. as belonging to a "family" and can trace their lineage from The folded three-dimensional comparisonsof their sequences. structuresof homologous proteins are similar evenif parts of their primary structure show little evidenceof homology. Initially, proteins with relatively high sequencesimilarities (>50 percent exact matches,or "identities") and related functions or structureswere defined as an evolutionarily related family, while a swperfamilyencompassedtwo or more families in which the interfamily sequencesmatched lesswell (=30-40 percent identities)than within one family. It is generally thought that proteins with 30 percent sequenceidentity are likely to have similar three-dimensionalstructures; however, proteins with far less sequencematching can have very similar structures.Recently,revised definitions of family and superfamily have been proposed, in which a family comprisesproteins with a clear evolutionary relationship (>30 percentidentity or additional structural and functional information showing common descent but <30 percent identity), while a superfamily comprisesproteins with only a probable common evolutionary origin (e.g., lower percent sequenceidentities).Often investigatorsconsider proteins to constitute a common superfamily (have a common evolutionary origin) when they contain one or more common motifs or domains. The kinship among homologous proteins is most easily visualizedby a tree diagrambasedon sequenceanalyses.For

example, the amino acid sequencesof globins, the proteins of hemoglobin and myoglobin and their relatives from bacteria, plJnts, and animals, suggestthat they evolved from an a.r.esiral monomeric, oxygen-binding protein (Figure 3-13)' With the passageof time, the gene for this ancestralprotein slowly changed, initially diverging into lineagesleading to animal and plant globins. Subsequentchangesgave rise to myoglobin. the monomeric oxygen-storingprotein in musc1., ind to the a and B subunits of the tetrameric hemoglobin molecule (cr2B2)of the circulatory system'

HierarchicalStructure of Proteins r A protein is a linear polymer of amino.acids linked together by peptide bonds. Various, mostly noncovalent, interactions between amino acids in the linear sequence stabilizea protein's specificfolded three-dimensionalstructure, or conformation' r The cr helix, B strand and sheet' and B turn are the most prevalent elementsof protein secondarystructure' Sectndary structuresare stabilized by hydrogen bonds between atoms of the peptide backbone' r Protein tertiary structure resultsfrom hydrophobic interactions betweennonpolar side groups and hydrogen bonds between polar side groups and the polypeptide backbone' EF P R O T E I N S . H I E R A R C H I C ASLT R U C T U RO

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These interactions stabilize folding of the secondarystructure into a compact overall arrangement. r Certain combinations of secondarystructuresgive rise to different motifs, which are found in a variety of pioteins and are often associatedwith specificfunctions (seeFigure 3-9). Proteins often contain distinct domains, independently Ided regions of secondaryor tertiary structure *ith .h.racteristicstructural, functional, and topological properties ( s e eF i g u r e3 - 1 0 ) . r The incorporation of domains as modules in different proteins in the course of evolution has generateddiversity in protein structure and function. r The number and organizationof individual polypeptide subunits in multimeric proteins define their quaternary structure. r Cellscontain largemacromolecularassemblies in which all the_necessary participantsin complex cellular processes(e.g., DNA, RNA, and protein synthesis;photosynthesis;signal transduction)are integratedto form molecular machines. Homologous proteins, which have similar sequences, ructures, and functions, evolved from a common ances_ tor. They can be classifiedinto families and superfamilies.

ProteinFolding

f[

As noted above,when it comesto biologicalstructuressuch as proteins, "form follows function" and .,form is function." Thus it is essentialthat when a polypeptideis synthesizedwith its particular primary structure (sequence),it folds into the

tricaciesof translation are consideredin Chapter 4. Here, we describethe key determinantsof the proper folding of a'nas_ cent (newly formed or forming) polypeptide chain.

P l a n a rP e p t i d eB o n d sL i m i tt h e S h a p e si n t o Which ProteinsCan Fold A critical structural feature of polypeptides that limits how the chain can fold is the planar p.piid. bond. Figure 3_3 il_ lustratesthe amide group in peptide bonds in a folypeptide chain. Becausethe peptide bond itself behave,puni"lty tit. a double bond, OO-

llt Pi"\1-P2 I a-a.

<-> r--u-':fi-P,

HH

the carbonyl carbon and amide nitrogen and those atoms directly bonded to them must all lie rn a fixed plane (Fig_ 74

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A FIGURE 3-14 Rotationbetweenplanarpeptidegroupsin proteins.Rotation aboutthe Co-amino nitrogen bond(the@angle) andthe C"-carbonyl carbonbond(thery'angle)permits polypeptide backbones, in principle, to adopta verylargenumberof potential conformations However stericrestraints dueto thestructure of the polypeptide backbone andthe properties of theaminoacidside chainsdramatically restrict the potential conformations thatcanbe adoptedby anygivenprotein ure 3-14); there is no rotation possibleabout the peptide bond itself. As a consequence,the only flexibility in a polypeptide chain backbone, allowing it to adopt varying conformations (twists and turns to fold into different threedimensional shapes),is rotation of the fixed planes of peptide bonds with respectto one another about two bonJsthe C.-amino nitrogen bond (rotational angle called d) and the C"-carbonyl carbon bond (rotational angle caled r/). Yet a further constraint on the potential conformations that a polypeptide backbone chain can adopt is that onlv a limited number of $ and ry'angles porribl., b..",rr" io. ".. most d and t! angles the backbone or side chain atoms would come too closeto one another and thus the associated conformation would be highly unstable or even physically impossibleto achieve.

Information Directinga protein,sFoldingls E n c o d e di n l t s A m i n o A c i d S e q u e n c e While the consrraintsof backbonebond anglesseemvery restrictive, any polypeptide chain containing only a few residuescould, in principle,still fold inro many conformations. For example,if the @and ry'angleswere limited to only eight combinations, an a-residue-longpeptide would potentially have 8" conformations-a very large number for evena small polypeptide of only 10 residueslong (about g.6 million possibleconformations)! In general,however,any particular protein adopts only one or just a few very closely related characteristic functional conformations called the natiue state; for the vast majority of proteins, the native state is the most stably folded form of the molecule. In thermodynamic terms, the native state is usually the conformation with the lowest free energy. lfhat features of proteins limit their folding from very many conformationi to iust one? The propertiesof the sidechains (e.g.,size,hydrophobicitg ability

to form hydrogen and ionic bonds)' together with their particular sequencealong the polypeptide backbone,impose key restrictions. For example, a large side chain such as that of tryptophan might prevent (stericallyblock) one region of the chain from packing closelyagainstanother region, whereasa side chain with a positive charge such as arginine might attract a segmentof the polypeptide that has a complementary negatively charged side chain (e.g., aspartic acid). Another example we have already discussed is the effect of the aliphatic side chains in heptad repeatson the formation of coiled coils. Thus, a polypeptide's primary structure determines its secondary,tertiary, and quaternary structure. The initial evidencethat the information necessaryfor a protein to fold properly is encodedin its sequencecame from in vitro studieson the refolding of purified proteins. Various perturbations (such as thermal energy from heat' extremes of pH that alter the chargeson amino acid side chains, and chemicals, called denaturants, such as urea or guanidine hydrochloride at concentrations of 6-8 M) can disrupt the weak noncovalent interactions that stabilize the native conformation of a protein, leading to its denaturation. Treatment with the reducing agents)such as B-mercaptoethanol, that break disulfide bonds can further destabilizedisulfidecontaining proteins. Under such unfolding or denaturing conditions, entropy increaseswhen a population of uniformly folded moleculesis destabilizedand converted into a collection of many unfolded, or denatured, molecules that have many different non-native and biologically inactive conformations. As we have seen,there are very many possible non-nativeconformations(e.g.,8"-1). The spontaneousunfolding of proteins under denaturing conditions is not surprising,given the substantialincreasein entropy. What is striking, however,is that when a pure sample of a singletype of unfolded protein is shifted back to normal conditions (body temperature' normal pH levels,reduction in the concentration of denaturantsby dilution or their removal), some denatured polypeptides can spontaneously renature (refold) into their native' biologically active states. This kind of refolding experiment, as well as studies that show synthetic proteins made chemically can fold properly, showed that sufficient information must be contained in the protein's primary sequenceto direct correct refolding. Newly synthesizedproteins appear to fold into their proper conformations just as denaturedproteins do. The observedsimilarity in the folded, three-dimensional structures of proteins noted in Section3.1, prowith similar amino acid sequences, vided additional evidencethat the primary sequencealso determinesprotein folding in vivo. It appearsthat formation of secondarystructuresand structural motifs occursearly in the folding process,followed by assemblyof more compact and complex domains, which then associateinto more complex tertiary and quaternary structures(Figure 3-15).

(c)

3-15 Hypotheticalprotein-foldingpathway'Folding A FIGURE of primary hierarchy proteinfollowsthestructural of a monomeric of small (b-d)+ tertiary(e)structureFormation (a)+ secondary of morestable formation to precede motifs(c)appears structural (e) (d)andthefinaltertiary structure domains

tide. However the conditions inside a cell are not the same as those in test tubes used for in vitro refolding experi-

Foldingof Proteinsin Vivo ls Promoted by Chaperones The refolding of a denatured protein is presumed to mimic many aspectsof the folding of a newly synthesizedpolypep-

vides. lfithout such help, they would waste much energy in p R o T E t NF 6 L D I N G

o

75

FocusAnimation:lhaperone-Mediated Foldingfllt ; ibosome

U n f o l d e dp r o t e i n N u c l e o t i d e - b i n d i ndgo m a i n UT

E GrpE/BAG1

\a

< FIGURE 3-16 Chaperone-mediated protein folding.Manyproteins foldintotheirproperthreedimensional structures with theassistance of Hsp70-like proteinsThesemolecular chaperones transiently bind to a nascent polypeptide asit emerges froma ribosome or to proteins thathaveotherwise unfoldedInthe Hsp70cycle,a substrate proteinbindsin rapid unfolded equilibrium to theopenconformation of the substratebindingdomain(SBD) of Hsp7O, to whichan ATpis boundin the nucleotide-binding (s1sp domain(NBD) a; proteins (DnaJ/Hsp4O) Accessory stimulate the hydrolysis of ATPandconformational changein Hsp70,resulting in theclosed form,in whichthesubstrate islockedinro the SBD;hereproperfoldingisfacilitated (stepZ) Exchange of ATPfor the boundADp,stimulated by otheraccessory (GrpE/BAG1), proteins converts the Hsp70backto the openform(stepE), releasing the properly (step4) foldedsubstrate

ATP ADP

the synthesisof improperly folded, nonfuncrional proteins. which would have to be destroyedto prevenrtheir disrupt_ ing cell function. Cells clearly have such mechanisms,since more than 95 percent of the proteins present within cells have been shown to be in their native ionformations. The explanation for the cell's remarkableefficiencyrn Dromot_ ing the proper protein folding encodedin primary *orr.r.. is that cells make a ser of proteins, called chaperon.r, th"t facilitate protein folding. The importance of chaperonesis highlightedby the observationsthat they are evolutionarily conserved,they are found in all organismsfrom bacteriato humans, and some are highly homologous and use almost identical mechanisms to assist p.ot.in folding. Chaper_ ones, which in eukaryotes are located in every cellular compartment and organelle, bind to rhe target proteins whose folding they will assist. Two general-families of chaperonea s re recognized:

to the exposureof hydrophobic side chains that have not yet had a chanceto be buried in the inner core ofthe folded piotein. These exposed hydrophobic side chains on diffeient moleculeswill stick to one another owing to the hydrophobic effect (Chapter 2) and thus promote aggregation.'Vfh.r, a newly synthesizedmolecule begins to fold, it is at risk of

Molecular Chaperones The heat-shockprotein Hsp70 and its homologs (Hsp70 in the cytosol and mitochondrial matrix, BiP in the endoplasmicreticulum, and DnaK in bac_

r Molecular chaperones,which bind and stabilize unfolded or partly folded proteins, thereby preventing theseproteins from aggregatingand being degraded r Chaperonins,which form a small folding chamber into which an unfolded protein can be sequesteied, giving it time and an appropriate environment to fold p-p..ly One reasonthat chaperonesare neededfor intracellular pro_ tein folding is that they help prevent aggregationof unfoided proteins. Unfolded and partly folded proteins tend to aggre_ gate into large, often water insoluble masses,from which it is extremely difficult for a protein to dissociateand then fold into its proper conformation. In part this aggregationis due

76

C H A P T E R3

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p R o T E t NS T R U C T U RAEN D F U N C T T O N

causesa conformational change in the chaperone rhat re_ leasesthe target protein. Additional proteins, such as the co-chaperoneHsp40 in eukaryotes (DnaJ in bacteria), help increise efficiency of

Hsp70-mediatedfolding of many proteins by stimulating the hydrolysis of ATP by Hsp70/DnaK (seeFigure 3-15). An additional protein called GrpE in bacteria (similar activity of BAG1 in mammals) also interacts with the Hsp70/DnaK, promoting the exchange of ATP for ADP. Multiple molecular chaperonesare thought to bind all nascent polypeptide chains as they are being synthesizedon ribosomes.In bacteria, 85 percent of the proteins are releasedfrom their chaperonesand proceedto fold normally; an even higher percentageof proteins in eukaryotesfollow this pathway. Chaperonins The proper folding of a large variety of newly synthesizedproteins also requires the assistanceof another class of proteins, the chaperonins. These huge cylindrical macromolecular assembliesare formed from two rings of oligomers, which can exist in a "tight" peptidebinding state and a "relaxed" peptide-releasingstate. The eukaryotic chaperonin TriC consistsof eight subunits per ring. In the bacterial, mitochondrial, and chloroplast chaperonin, known as GroEL, each ring contains sevenidentical subunits (Figure 3-1'7a).The GroEL folding mechanism, which is better understood than TriC-mediated folding, servesas a general model (Figure 3-1'7b).A partly folded or misfolded polypeptide is inserted into the cavity of the barrel-like GroEL. where it binds to the inner wall and folds into its native conformation. In an MP-dependent step, GroEL undergoesa conformational change and releasesthe folded protein, a processassistedby a co-chaperonin, GroES, which caps the ends of GroEL. The binding of ATP and the co-chaperoninGroES to one of the rings in the tight state of GroEL causesa twofold expansion of its cavity, shifting the equilibrium toward the relaxedpeptidefolding state. There is a striking similarity between the capped-barreldesign of GroELiGroES, in which proteins are sequesteredfor folding, and the structure of the 265

proteasomethat participatesin protein degradation (discussedin Section3.4).

AlternativelyFoldedProteinsAre lmplicated in Diseases As noted earlier, each protein normally folds into a sin-

Eil gl., energeticallyfavorableconformationthat is speciRecentevidencesuggests' fied by its amino acid sequence. however, that a protein may fold into an alternative threedimensional structure as the result of mutations, inappropriate covalent modifications made after the protein is synthesized,or other as-yet-unidentifiedreasons.Such "misfolding" not only leads to a loss of the normal function of the protein but often marks it for proteolytic degradation. However, when degradation isn't complete or doesn't keep pace with misfolding, the subsequentaccumulation of the misfolded protein or its proteolytic fragments contributes to certain degenerative diseases characterizedby the presenceof insoluble protein plaques in various organs, including the liver and brain. Some neurodegenerativediseases,including Alzheimer's diseaseand Parkinson'sdiseasein humans and transmissible

ments or the soluble alternativelyfolded proteins are toxic to the cell is unclear. I

ffi viu"o:GroEl Pasecycle (b) Ribosome

Partiallyfolded or misfoldedprotein

proteinfolding.Proper 3-17 Chaperonin-mediated A FIGURE suchasthe on chaperonins depends foldingof someproteins of 14 complex isa hollow,barrel-shaped prokaryotic GroEL(a)GroEL (b) In rings in two stacked arranged subunits 60,000-MW identical in a "tight" exists of ADBGroEL of ATPor presence the absence proteins. statethatbindspartlyfoldedor misfolded conformational

Protein

Properly folded protein

to a moreopen,"relaxed"state,which Bindingof ATPshiftsGroEL is oneendof GroEL Duringthisprocess, thefoldedprotein. releases of assembly an GroES, bytheco-chaperonin blocked transiently Cell87:241' (a)fromA Roseman etal, 1996' subunits[Part 1O,OO0-MW Saibil H of l courtesy

P R O T E I NF O L D I N G

77

(a)

j r Some neurodegenerativediseasesare causedby aggrej S"t."r of proteins that are s r a b l y f o l d e d i n a n a l t e r n a r i v e conlormatlon. I

ff,l

ProteinFunction

Although proteins have many different shapesand sizesand mediate an extraordinarily diverse array of activitiesboth inside and outside of cells, most of thesediversefunctions are basedon rheabilityof proteinsro engagein a commonactiviry, the binding to themselves,other macromolecules,small mole, cules,and ions. Here we will describesomeof the key features underlyingprotein binding, and then turn to look at one group of proteins, enzymes,in greater detail. The activities of the other functional classesof proteins (structural,scaffold,transport, regulatory,motor) will be describedin other chapters.

S p e c i f i cB i n d i n go f L i g a n d sU n d e r l i e st h e F u n c t i o n so f M o s t P r o t e i n s

-*si

-:* t2oPtt

,

100nm

,

FIGURE3-18 Alzheimer,sdiseaseis characterizedby the formation of insoluble plaques composed of amyloid protein. ( a )A t l o w r e s o l u t i o na ,n a m y l o i dp l a q u ei n t h e b r a i no f a n Alzheimers patientappearsas a tangleof filaments.(b)The reqularstructureof f rom p.aquesrsrevealedrn rhe atomicforcemicroscope. frlaments Proteolysis of the naturallyoccurringamyloidprecursorproteinyields a shortfragment,cailedp-amyloidprotern,that for unKnownreasons changesfrom an a-helicalto a B-sheetconformationThisalternative

degenerative diseases[Courtesv of K Kosk I

The molecule to which a protein binds is often calleclits ligand. In some casesligand binding causesa changein the shape of a protein. Ligand-binding-drivenconformational changesare integral to the mechanismof action of many proteins and are important in regulating protein activity. Two propertiesof a protein characterizehow it binds ligands.Specificity refersto the ability of a protein to bind one

ure of affinity (Chapter 2). The stronger the interaction be-

P r o t e i nF o l d i n g r The sequence of a proteindeterrnines its three-dimensional structure, which detern.rines its function. In short, function derivesfrom structure;structurederivesfrom sequence. Becauseprotein function derivesfrom protern srructure, wly synthesized proteinsmust fold into the correctshaoe f u n c r i o np r o p e r l y . r The planar structureof the peptidebond limits the num_ ber of conformationsa polypeptidecan have. The amino acid sequenceof a protein dictatesits foldine to a specificthree-dimensional conformarion.rhe native state. Proteins will unfold, or denature,if treated under conditionsthat disrupt rhe noncovalenrinteractionsstabi_ lizing their three-dimensional structures. olding in vivo occurswith assistance from chaoich bind ro nascentpolypeptidesemergingfrom and prevenrtheir misfoldine. 78

.

cHAprER 3

I

p R o r E l Ns r R U c r u R A E N DF U N c l o N

virus) or other foreign substances(e.g.,proteinsor polysaccha_ ridesin pollens).Different antibodiesare generatedin response to differentanrigens,and theseantibodieshavethe remarkable characteristicof binding specificallyto (..recognizing,')a por_ tion of the antigen,called an epitope,which initially induced the production of the antibody, and not to other molecules. Antibodies act as specificsensorsfor antigens,forming antibody-antigen complexesthat initiate a cascadeof proiective r e e c t i o nisn c e l l so f t h e i m m u n es y s r e m .

CDR

Light chain lnterchain disulfide bonds

"t"

Heavychain

Carbohydrate

periments discussedin subsequentchapters' \7e will see many examples of protein-ligand binding throughout this book, including hormones binding to receptors (Chapter 15), regulatory moleculesbinding to DNA (Chapt , i\. cell-adhesion molecules binding to extracellular matrix (Chapter 1'9),to name just a few. Next we will consider how the binding of one class of proteins, enzymes,to their ligands results in the catalysisof the chemical reactions essentialfor the survival and function of cells.

EnzymesAre HighlYEfficientand SpecificCatalysts Proteins that catalyze chemical reactions, the making and

3-19 Protein-ligandbindingof antibodies.(a)Ribbon A FIGURE molecule of the immunoglobulin Every antibody modelof an antibody. heavychains(lightanddarkred)and of two identical lgGclassconsists bondsThe linkedby disulfide lightchains(blue)covalently two rdentical the tvuoheavy containlng the overall structure diagram of insetshowsa and fit betweenan antibody andtwo lightchains(b)Thehand-in-glove on itstargetantrgen-inthiscase, thesiteto whichit binds(epitope) make Regions wherethetwo molecules lysozyme egg-white chicken contacts theantigenwith Theantibody contactareshownassudaces. (CDRs). Inthis regions fromallitscomplementarity-determining residues of theantigenandantibodyis complementarity view,the molecular fromtheantigensurface where"fingers"extending apparent especially surface. to "clefts"in theantibody areopposed

catalyzed by a specific efizyme. In many ways' enzymes are the cell's chemists,performing many of a cell's chemical reactions. (An additional form of catalytic macromolecule in cells is made from RNA. TheseRNAs are called ribozymes') Thousands of different types of enzymes'each of which catalyzesa single chemical reaction or set of closely related reactions,have beenidentified. Certain enzymesare found in the majority of cells becausethey catalyze the synthesisof common cellular products (e.g.,proteins, nucleic acids, and phospholipids) or take part in the production of energy (e'g'' ty th. .ottu.rsion of glucoseand oxygen into carbon dioxide and water). Other enzymesare present only in a particular

tract. or even outside the organism (e.g., toxic enzymesin the venom of poisonous snakes). Like all catalysts,enzymesincreasethe rate of a reaction but do not affect the extent of a reaction, which is deter-

AII antibodies are Y-shapedmoleculesformed from two identical heavy chains and two identical light chains (Figure 3-19a). Each arm of an antibody moleculecontains a singlelight chain linked to a heavy chain by a disulfide bond. Near the end of each arm are six highly variable loops' called complementarity-determiningregions (CDRs/, which form the antigen-binding sites.The sequencesof the six loops are highly variable among antibodies, generating unique complementary ligand-binding sites that make them specific for different epitopes (Figure 3-19b). The intimate contact between thesetwo surfaces,stabilizedby numerousnoncovalent interactions, is responsiblefor the extremely precisebinding specificity exhibited by an antibody. P R O T E I NF U N C T I O N

O

79

-__Transitionstate (uncatalyzed)

t

0) q) o o '-I

(a) Catalyticsite

(b)

Catalyticsite

Binding pocket

Transitionstate (catalyzed)

. A/l.+ ."cat

B i n d i n gp o c k e t

Products

Progressof reaction-----> A FIGURE 3-20 Effectof an enzymeon the activationenergy of a chemicalreaction.Thishypothetical reaction pathwaydepicts the changes in freeenergyG asa reaction proceeds A reaction will takeplacespontaneously onlyif the totalG of the products isless thanthatof the reactants (negative AG) However, allchemical reactions proceed throughoneor morehigh-energy transition states, andthe rateof a reaction isinversely proportional to the activation energy(AG+), whichisthedifference in freeenergybetween the reactants andthetransition state(highest pointalongthe pathway). Enzymes andothercatalysts accelerate the rateof a reactionby reducing thefreeenergyof thetransition stateandthusAG+. rates of reactions 106-1012times that of the corresponding uncatalyzedreactions under otherwise similar conditions.

An Enzyme'sActive Site BindsSubstrates a n d C a r r i e sO u t C a t a l y s i s

sitesmake up only a small fraction of the total protein, with the rest involved in folding of the polypeptide, iegulation of the active site, and interactions with other molecules.

A FIGURE 3-21 Active site of the enzymetrypsin. (a)An enzyme's activesiteiscomposed of a bindingpocket, whichbinds specifically to a substrate, anda catalytic site,whichcarnes out catalysis(b)A surfacerepresentation of the serineprotease trypsin Activesitecleftscontaining thecatalytic site(sidechains of the catalytic triadSer-195, Asp-102, andHis-57 shownasstickfigures) andthe substrate sidechainspecificity bindingpocketareclearly visible[Part(b)courtesy of PTeesdale-spittte ]

substrate-bindingsite and the substrate,which is mediated by multiple weak noncovalent interactions and is very sensitive to the shapesof substrates.Usually only one or a few substratescan fit preciselyinto a binding site. The idea that enzymesmight function by binding to rheir substratesin the manner of a key fitting into a lock was suggestedfirst by Emil Fischerin1894.Ln1913 Leonor Michaelis and Maud Leonora Menten provided crucial evidence supporting this hypothesis.They showed that the rate of an enzymatic reaction was proportional to the substrateconcentration at low substrateconcentrations, but that as the substrateconcentrations increased,the rate reached a maximal velocity V-o and became substrate concentration-independent, with the value of V-"" being directly proportional to the amount of enzymepresent in the reaction mixture (Ftgure 3-22). They deducedthat this saturation at hieh substratecon-

termediate step in the ultimately irreversible conversion of substrateto product (P) (Figure3-23): substratehas bound. In some enzymes,the catalytic and substrate-binding sites overlap; in others, the two regions are structurally as well as functionally distinct.

E + S i------^ES-+E + p and that the rate Ve of formation of product at a particular substrateconcentration [S] is given by what is now called the Mich aelis-Mentenequahon:

vo:v-"*;J!+ Km L)l

zyme. As noted above, this specificity of enzymesis a conse_ quenceof the precisemolecular complementarity betweenits 80

o

c H A p r E 3R | p R o r E t N s r R u c r u RA EN DF U N c l o N

(3-1)

where the Michaelis constant K-, a measureof the affinity of an enzyme for its substrate (seeFigure 3-22), is the substrate concentration that yields a half,maximal reaction rate

(a) c .=6

2.0 oJ

b.> t.5 ^=

. : : J /

6-

E(L

1.0

Enzyme

0.5 o:9 t

c().

Concentrationof substrateIS]

f I

etnoino no.t"t

(b)

1.0 o nR

I 0.6

High-affinity substrate L o w - a f fi n i t y s u b s t r a t e( S ' )

I

g 0.4 o

o.2 0

(tSlor tS'l) of substrate Concentration

reaction' 3-22 K^ and V."* for an enzyme-catalyzed A FfGURE of the of the dependence fromanalysis K, andVru"aredetermined Theshapeof concentration velocity on substrate initialreaction of a simpleenzymeischaracteristic kineticcurves thesehypothetical (S)isconverted into in whichonesubstrate reaction catalyzed after immediately product(P)Theinitralvelocity is measured concentration to substrate beforethe substrate additionof enzyme (a)Plotsof the initialvelocrty at two different appreciably changes concentration of enzyme concentrations [E]asa functionof substrate Michaelis rate is the yields reaction half-maximal a The that lsl [S] of theaffinityof Efor turningS intoP K., a measure constant increase a proportronal causes concentration theenzyme Quadrupling quadrupled; is V.u" velocity the maximal rate,andso in the reaction (b)Plotsof the initialvelocity versus isunaltered. theK., however, hasa Sfor whichtheenzyme witha substrate concentration substrate hasa lower S' for whichtheenzyme highaffinityandwitha substrate because affinity.Notethatthe V.u"isthe samewith bothsubstrates, substrate low-affinity for S', the Km is higher that IE]isthesame,but

(i.e.,112V-.*), and thus is analogousto the dissociationconstant K6 (Chapter 2). The smaller the value of K-, the more effectivethe enzyme is at making product from dilute solutions of substrateand the smaller the substrateconcentration neededto reach half-maximal velocity.The concentrationsof the various small moleculesin a cell vary widely, as do the Kvaluesfor the different enzymesthat act on them. A good rule of thumb is that the intracellular concentrationof a substrate is approximately the same as or somewhat greater than the K^ value of the enzymeto which it binds' The rates of reaction at substratesaturation vary enormously among enzymes.The maximum number of substrate moleculesconvertedto product at a singleenzymeactivesiteper secondis called the turnouer nwmber,which can be lessthan 1

Enzyme

3-23 Schematicmodel of an enzyme'sreaction A FIGURE (E)bind thatenzymes suggest kinetics Enzyme mechanism. (5)througha fixedandlimitednumberof sites molecules substrate tsknownasan (theactivesites). Theboundspecies on theenzymes with in equilibrium is (ES) ES complex The complex. enzyme-substrate stepin andisan intermediate andsubstrate the unboundenzyme to products(P) of substrate the conversion

substrate complexes (ES' ES', ES", etc.) generatedprior to the final releaseof the Products: E+S

.

^

--^ ES' ^ S' E S F . . . . .E

'"'E+P

The energyprofiles for such multistep reactionsinvolve multiple hills and valleys (Figure 3-24), and methods have been developedto trap the intermediatesin such reactionsto learn more about the details of how enzymescatalyzereactions'

SerineProteasesDemonstrateHow an Enzyme's Active Site Works Serineproteases,a large family of proteolytic enzymes' are used throughout the biological world-to digest meals (the FUNCTION PROTEIN

81

(a)

(b)

()

(5

Enzyme-transition statecomplex

j

6 o o

o) q) o o) LI

0) IL

EX+ E n z y m e+ substrate

Enzymesubstrate comprex

E+S E+P

Progressof reaction-----> S<-X++P A FIGURE 3-24 Free-energyreactionprofilesof uncatalyzed and multistepenzyme-catalyzed reactions.(a)Thefree-energy profileof a hypothetical reaction simpleuncatalyzed reaction converting (S)to product(p)viaa singlehigh_energy substrate transition state.(b)Manyenzymes catalyze suchreactions by dividing the process intomultiple discrete steps,in thiscasethe initial pancreatic enzymestrypsin, chymotrypsin, and elastase),to

consider how trypsin and its two evolutionarily closely related pancreatic proteases,chymotrypsin and elastase,catalyze cleavageof a peptide bond:

o

Progressof reaction-----> E+S*ES+EX+------+E+p formation of an EScomplex followedby conversion viaa single transition state(EX+) (E)andp Theacrrvatron to thefreeenzyme energyfor eachof thesestepsissignificantly lessthanthe activation energyfor the uncatalyzed reaction; thustheenzyme dramatically enhances the reaction rate. Figure 3-25a shows how a substratepolypeptide binds to the substrate-bindingsite in the active site of trypsin. There are two key binding interactions.First, the substrateand enzyme form hydrogen bonds that resemblea B sheet.Second, a key side chain of the substratethat determineswhich peptide in the substrate is to be cleaved extends into the enzyme's side-chain-specificitybinding pocket, at the bottom of which resides the negatively charged side chain of the (a)

x ,9

-:'

p(""'"tr1-P2 + HrO

p,,-C...-

+

Catalyticsite

NH3+-P2

His-57

l*o

H

Ho'

\r'r

where P1is the portion of the protein on the N-terminal sideof the peptide bond, and P2is the portion on rhe C-terminalside. S7efirst consider how serineproreasesbind specificallyto their substratesand then show in detail how catalysistakesplace.

C: O-

82

.

c H A p r E R3

|

p R o r E t Ns r R U c r u R EA N D F U N c l o N

Trypsin

Oxyanion hole

A r g i n i n es i d e c h a i n( R 3 )i n substrate

> FIGURE 3-25 Substratebindingin the activesite of typsin_ N like serineproteases. (a)Theactivesiteof trypsin(bluemolecule) H wrtha boundsubstrate (blackmolecule) Thesubstrate formsa two_ B i n d i n gs i t e stranded B sheetwith the bindingsite,andthe sidechainof an (Rr)in thesubstrate arginine is boundin the side-chain-specificitv binding p o c k e tl .t sp o s i t i v ecl h y a r g egdu a n i d i n i ugmr o u pi s stabilized by the negative chargeon the sidechainof the enzyme,s A s p - 1 8 9T h i sb i n d i n g a l i g n tsh ep e p t i d b e o n do f t h ea r g i n i n e appropriately (b) for hydrolysis catalyzed by theenzyme,s active_site c a t a l y tti rci a d( s i d ec h a i nos f S e r - 1 9 5H,i s - 5 a7 n dA s p - 1 0 2()b. )T h e a m i n oa c i d sl i n i n gt h es i d e - c h a i n - s p e c ibf i n c idt iyn g pocket d e t e r m i ni e t ss h a p ea n dc h a r g ea,n dt h u si t sb i n d i n g properties Trypsin accommodates the positively charged sidechainsof arginine and lysine; chymotrypsin, large,hydrophobic sidechainssuchas p h e n y l a l a n ianned; e l a s t a ssem , a lsl r d ec h a i nssu c ha sg l y c i naen d

alanine.[Part(a)modified from.J.J. perona andC. S. Craik,1991. J. Biol. Chen 272(48).29987-29990 I

Peptidebond to be cleaved

Side-chainspecificity binding pocket Asp-189

Chymotrypsin

Elastase

enzyme'sAsp-189. Trypsin has a marked preferencefor hydrolyzing proteins (black in Figure 3-25a) at the carboxyl side of a residue with a long positively charged side chain (arginine or lysine), becausethe side chain is stabilizedin the specificity binding pocket by the negativeAsp-189. Slight differencesin the structures of otherwise similar specificity pockets help explain the differing substratespecificities of the two related serine proteases:chymotrypsin prefers large aromatic groups (asin Phe,Tyr, Trp), and elastaseprefers the small side chains of Gly and Ala (Figure 3-25b1.The unchargedSer-189in chymotrypsinallows large, uncharged,hydrophobic side chains to bind stably in the pocket. The branchedaliphatic side chainsof valine and threoninein elastase replace glycines in the sides of the pocket in trypsin and thus prevent large side chains in substratesfrom binding, but allow stablebinding of the short alanineor glycinesidechain. In the catalytic site, all three enzymesuse the hydroxyl group on the side chain of a serine in position 195 to catalyzethe hydrolysis ofpeptide bonds in protein substrates.A catalytic triad formed by the three side chains of Ser-195, His-57, Asp-102 participates in what is essentiallya twostep reaction. Figure 3-26 shows how the catalytic triad co-

operates in breaking the peptide bond, with Asp-102 and His-57 supporting the attack of the hydroxyl oxygen of Ser-195 on the carbonyl carbon in the substrate.This attack initially forms an unstable transition state with four groups attached to this carbon (tetrahedral intermediate). Breaking of the C-N peptide bond then releasesone part of the protein (NH3-P2), while the other part remains covalently attached to the enzymevia an ester bond to the serine'soxygen, forming a relatively stable intermediate (the acyl enzyme). The subsequentreplacementof this oxygen by one from water, in a reaction involving another unstable tetrahedral intermediate, leadsto releaseof the final product (P1-COOH). The tetrahedral intermediates are partially stabilized by hydrogen bonding from the enzyme'sbackbone amino groups in what is called the oxyanion hole.The large family of serine proteasesand related enzymeswith an active-siteserine illustrates how an efficient reaction mechanism is used over and over by distinct enzymesto catalyzesimilar reactions. The serineproteasemechanismpoints out severalgeneral key featuresof enzymaticcatalysis:(1) enzymecatalytic sites are designedto stabilizethe binding of a transition state' thus lowering the activation energy and acceleratingthe overall

(c) Acyl enzyme (ES'complex)

(b) Tetrahedralintermediate (transitionstate)

( a ) E Sc o m p l e x

o H 'r'r.,..-N

D , rH; N

"c

/ Oxyanion hole

P.,/

P,,, H

T,o (f) EP complex

(d) Acyl enzyme (ES'complex)

(e) Tetrahedralintermediate (transitionstate)

,,\ _ . N

HN.

\J

A FIGURE 3-26 Mechanismof serineprotease-mediated His-57, triadof Ser-195, hydrolysisof peptidebonds.Thecatalytic proteases employs a multistep in theactive sitesof serine andAsp-102 peptidebondsin targetproteins(a)Aftera mechanism to hydrolyze polypeptide substrate bindsto the activesite(seeFigure3-23)forming the carbonyl 95 attacks oxygenof Ser-1 the hydroxyl an EScomplex, targetedpeptidebond(yellow)Movements carbonof the substrate's in the areindicated by arrows.(b)Thisattackresults of electrons in intermediate, statecalledlhe tetrahedral formationof a transition isstabilized by oxygen on thesubstrates charge whichthenegative hole.(c)Additional s oxyanion hydrogen bondsformedwiththe enzyme of the peptidebond,release resultin the breaking electronmovements

\-

,,,.\

_ HN. ,_-NH

\l

(lowpH) Inactive Active (NH2-P2), products andformationof the acyl of oneof the reaction watermolecule enzyme(ES'complex)(d)An oxygenfroma solvent (e)Thisattack enzyme the acyl of carbon the carbonyl thenattacks intermediate tetrahedral of a second in theformation results of the Ser(fl Additional resultin the breaking electronmovements of the andrelease of the EPcomplex) bond(formation 195-substrate is which (P1-COOH). of His-57, chain The side product finalreaction the side chain to bonding hydrogen by proper orientation heldin the protons anddonating bywithdrawing catalysis facilitates of Asp-102, (inset)lf the pH istoo low andthe sidechain the reaction throughout andthe in catalysis it cannotparticipate of His-57isprotonated, isinactive enzyme FUNCTION PROTEIN

83

reaction, (2) multiple side chains, togerherwith the polypeptide backbone,carefully organizedin three dimensions,work togetherto chemicallytransform substrateinto product, often by multistepreactions,and (3) acid-basecatalysismediatedby one or more amino acid side chainsis often used by enzymes, as when the imidazolegroup of His-57 in serineproteasesacts as a base to remove the hydrogen from Ser-195,shydroxyl group. As a consequence,often only a particular ionization state (protonated or nonprotonated) of one or more amrno acid side chains in the catalytic site is compatible with catalysis,and thus the enzyme'sactivity is pH-dependent. For example,the imidazoleof His-57 in serineproreases, whose pK" is =5.8, can help the Ser-195hydroxyl attack the substrateonly if it is not protonated. Thus, the activity of the proteaseis low at pH < 6.8, and the shapeof the pH activity profile in the pH range 4-8 matches the titration of the His-57 side chain, which is governed by the HendersonHasselbalchequation,with an inflection near pH 6.8 (seeFigure 3-27, right, and Chapter 2). The activity drops at higher pH values,generatinga bell-shapedcurve, becausethe proper folding of the protein is disrupted when the amrno group ar the protein's amino rerminus is deprotonated (pK" = 9); the conformation near the active site changesas a consequence. The pH sensitivity of an enzyme'sactivity can be due to changesin the ionization of catalytic groups, groups that participate directly in substratebinding, or groups that influence the conformation of the protein. Pancreaticproteasesevolved to function in the neutral or slightly basic conditions in the intestines;hence,their pH oprima are =8. proteasesand other

Lysosomalenzvme

o 0)

E N

c o) 0) G

o

2345678910 oH A FfGURE 3-27 pH dependence of enzymeactivity.lonizable (pH-titratable) groupsin theactivesitesor elsewhere in enzymes oftenmustbe eitherprotonated or deprotonated to permitproper substrate bindingor catalysis or to permitthe enzyme to adoptthe correctconformation Measurement of enzyme activity asa function of pHcanbe usedto identify the pKa'sof thesegroupsThe pancreatic serineproteases, (rightcurve), suchaschymotrypsin exhibitmaximum activity aroundpHg because of titrationof the (required activesiteHis-57 for catalysis, pKu= 6 8) andof theamino terminus of the proteln(required for properconformation, pK"= 9) Manylysosomal hydrolases haveevolved to exhibita lowerpH optimum(=45, leftcurve) to matchthe low internal pH in lysosomes in whichtheyfunction. fromp Lozano, [Adapted T.DeDiego, andJ L l b o r r a ,1 9 9 7 ,E u r J B i o c h e m2 4 8 ( 1) : 8 0 - 8 5 ,a n d W A J u d i c ee t a l , 2 0 0 4 . E u r . J B i o c h e m2 7 1 ( 5 ) : 1 0 4 6 - 1 0 5l3

84

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hydrolytic enzymesthat function in acidic conditions must employ a different catalytic mechanism.This is the casefor enzymeswithin the stomach (pH = 1) such as the protease pepsin or those within lysosomes(pH = 4.5), which play a key role in degrading macromoleculeswithin cells (seeFigure 3-27, left).Indeed, lysosomal hydrolasesthat degrade a wide variety of biomolecules(proteins, lipids, etc.) are relatively inactive at the pH in the cytosol (=7), and that helps protect a cell from self-digestionshould theseenzymesescape the confinesof the membrane-boundlysosome. One key feature of enzymatic catalysisnot seenin serine proteases,but found in many other enzymes,is a cofactor,or prosthetic (helper) group. This is a nonpolypeptide small molecule or ion (e.g., iron, zinc, copper, manganese)that is bound in the active site and plays an essentialrole in the reaction mechanism. Small organic prosthetic groups in enzymesare also caIIedcoenzymes.Someof theseare chemically modified during the reaction and thus need to be replaced or regeneratedafter each reaction; others are not. Examplesof the former include NAD* (nicotinamideadenine dinucleotide) and FAD (flavin adenine dinucleotide) (see Figure 2-33), whereas heme groups that bind oxygen in hemoglobin or transfer electrons in some cytochromes are examples of the latter (Figure 12-14). Thus, the chemistry catalyzedby enzymesis not resrrictedby the limited number of amino acids in polypeptide chains. Many vitamins-e.g., the B vitamins, thiamine (B1), riboflavin (B2), niacin (B3), and pyridoxine (86), and vitamin C-which cannot be synthesized in higher animal cells, function as or are used to generatecoenzymes.That is why supplementsof vitamins must be added to the liquid medium in which animals cells are grown in the laboratory (Chapter9). Small moleculesthat can bind to active sitesand disruot the reactionsare called enzymeinbibitors. Suchinhibitois are useful tools for studying the roles of enzymesin cells and whole organisms by allowing analysis of the consequencesof the loss of the enzyme'sactivity. Thus, inhibitors complement the use of mutations in genesfor probing an enzyme'sfunction in cells (seeChapter 5). However, interpreting results of inhibitor studies can be complicated if, as is often the case,rhe inhibitors block the activity of more than one protein. Smallmolecule inhibition of protein activity is the basis for mosr drugs (e.g., aspirin inhibits enzymescalled cyclooxygenases) and also for chemical warfare agents. Sarin and other nerve gasesreact with the active serinehydroxyl groups of both serine proteasesand a related enzym%acetylcholineesterase,which is a key enzymein regulating nerve conduction (seeChapter 23). I

Enzymesin a CommonPathwayAre Often PhysicallyAssociatedwith One Another Enzymes taking part in a common metabolic process (e.g., the degradation of glucoseto pyruvate) are generallylocated in the same cellular compartment (e.g., in the cytosol, at a membrane,or within a parricularorganelle).'Withina compartment, products from one reaction can move by diffusion to the next enzyme in the pathway. However, diffusion

(a)

Reactants

/

Reactants

Products

sized bacterium experiencesa drag force from water that stops its forward movement within a fraction of a nanometer when it stops actively swimming. To generatethe forces necessaryfor many cellular movements,cells dependon specialized enzymes commonly called molecular motors, or motor proteins. These mechanochemicalenzymes convert energyreleasedby the hydrolysis of ATP or contained within ion gradients into a mechanical force, usually generating either linear or rotary motion. From the observed activities of motor proteins' we can infer three generalproperties that they possess: r The ability to transducea source of energy'either ATP or an ion gradient, into linear or rotary movement

Products (c)

r The ability to bind and translocatealong a substrate r Net movement in a given direction \(/e will see many examples of such motors in subsequent chapters.

A FIGURE 3-28 Assemblyof enzymesinto efficient pathways reaction Inthe hypothetical multienzymecomplexes. intofinalproducts herethe initialreactants areconverted illustrated A, B,andC. (a)Whenthe actionof threeenzymes: bythe sequential withinthesame in even constrained are free solution or enzymes sequence in the reaction compartment, the intermediates cellular slow to the next,an inherently mustdiffusefromoneenzyme (b)Diffusion process. whenthe isgreatlyreduced or eliminated eitherby intomultisubunit complexes, enzymes associate individual protein(c)Theclosest or with the aidof a scaffold themselves whentheenzymes occurs of different catalytic activities integration in a single domains level,becoming arefusedat the genetic polypeptide chain

entailsrandom movement and can be a slow, relatively inefficient processfor moving moleculesbetweenwidely dispersed enzymes(Figure 3-28a). To overcomethis impediment, cells have evolved mechanismsfor bringing enzymesin a common pathway into closeproximity. In the simplest such mechanism, polypeptides with different catalytic activities cluster closely together as subunits of a multimeric enzyme or assembleon a common "scaffold" that holds them together(Figure3-28b). This arrangement allows the products of one reaction to be channeled directly to the next enzyme in the pathway. In some cases, independentproteins have been fused together at the genetic level to createa singlemultidomain, multifunctional enzyme (Figure3-28c).

E n z y m e sC a l l e dM o l e c u l a rM o t o r s C o n v e r t Energyinto Motion At the nanoscaleof cells and molecules,movement is influenced by forces that differ from those in the macroscopic world. For example, the high protein concentration (200300 mglml) of the cytoplasm preventsorganellesand vesicles from diffusing faster than 100 pm/3 hours. Even a micrometer-

Protein Function r The functions of nearly all proteins depend on their ability to bind other molecules(ligands). r The specificity of a protein for a particular ligand refers to the preferential binding of one or a few closely related ligands. r The affinity of a protein for a particular ligand refers to the strength of binding, usually expressedas the dissociation constant K6. r Ligand-binding sites on proteins and the corresponding ligands themselvesare chemically and spatially complementary. r Enzymes are catalyticproteins that acceleratethe rate of cellular reactions by lowering the activation energy and stabilizing transition-stateintermediates(seeFigure 3-20). r An enzyme active site, which is usually only a small part of the protein, comprisestwo functional parts: a substratebinding site and a catalytic site. The amino acids composing the active site are not necessarilyadjacent in the amino acid sequencebut are brought into proximity in the native conformation. r The substrate-bindingsite is responsiblefor the exquisite specificity of enzymesowing to its molecular complementarity with the substrateand the transition state. r The initial binding of substrates(S)to enzymes(E) results in the formation of an enzyme-substratecomplex (ES)' which then undergoesone or more reactions catalyzed by the catalytic groups in the active site until the products (P) are formed and diffuse away from the enzyme. r From plots of reaction rate versus substrate concentration, two characteristicparametersof an enzymecan be determined: the Michaelis constant K-, a rough measureof P R O T E I NF U N C T I O N

85

the enzyme'saffinity for converting substrateinto product, and the maximal velocity V*^*, d measureof its catalytic power (seeFigure 3-22). r The rates of enzyme-catalyzedreactions vary enormously, with the turnover numbers (number of substratemolecules converted to products at a singleactive site at substratesaturation) ranging between<1 to 6 x 10s molecules/s. r Many enzymes catalyzethe conversion of substratesto products by dividing the process into multiple discrete chemical reactions that involve multiple distinct enzyme substratecomplexes(ES',ES",etc.). r Serineproteaseshydrolyze peptide bonds in protein substratesusing as catalyticgroups the sidechainsof Ser-195, H i s - 5 7 ,a n d A s p - 1 0 2 . r Amino acids lining the specificity binding pocket in the binding site of serine proteasesdetermine the residue in a protein substrate that will be hydrolyzed and account for differencesin the specificity of trypsin, chymotrypsin, and elastase. r Enzymesoften use acid-basecatalysismediated by one or more amino acid side chains,such as the imidazolegroup of His-57 in serineproteases,to catalyzereactions. r The pH dependenceof protonation of catalytic groups (pK") is often reflected in the pH-rate profile of the enzyme's activity. The pH sensitivity of an enzyme'sactivity can be due to changesin the ionization of catalytic groups, of groups that participate directly in substrate binding, or of groups that influence the conformation of the protein. r In some enzymes,nonpolypeptide small moleculesor ions, called cofactors or prosthetic groups, can bind to the active site and play an essentialrole in enzymaric catalysis.Small organic prostheticgroups in enzymesare also called coenzymes;vitamins, which cannot be synthesized in higher animal cells, function as or are used to generate coenzymes. r Enzymesin a common pathway are located within specific cell compartments and may be further associatedas domains of a monomeric protein, subunits of a multimeric protein, or componentsof a protein complex assembledon a common scaffold(seeFigure 3-28). r Motor proteins are mechanochemicalenzymesthat convert energy releasedby ATP hydrolysis into either linear or rotary movement.

Regulating ProteinFunction l:

fll

ProteinDegradation Most processesin cells do not take place independently of one another or at a constant rare. The activities of all oroteins and other biomoleculesare regulated to integrate their functions for optimal performance for survival. For example, the catalytic activity of enzymesis regulated so that the amount of reaction product is lust sufficient to meet the 86

.

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needsof the cell. As a result, the steady-stateconcentrations of substratesand products will vary, depending on cellular conditions. Regulation of nonenzymaticproteins-the opening or closing of membrane channels or the assembly of a macromolecular complex, for example-is also essential. In general,there are three ways to regulateprotein activity. First, cells can increaseor decreasethe steady-statelevel of the protein by altering its rate of synthesis, its rate of degradation, or both. Second,cells can change the intrinsic activity, as distinct from the amounr, of the protein (e.g.,the affinity of substratebinding, the fraction of time the protein is in an active versus inactive conformation). Third. there can be a change in location or concentration within the cell of the protein itself, the target of the protein's activity (e.g., an enzyme'ssubstrate),or some other molecule required for the protein's activity (e.g., an enzyme'scofactor). All three types of regulation play essentialroles in the lives and functions of cells.

RegulatedSynthesisand Degradationof Proteinsls a FundamentalPropertyof Cells Controlof ProteinSynthesis The rateof synthesis of proteins is determined by the rate at which the DNA encoding the protein is converted to mRNA (transcription), the steady-stateamount of the active mRNA in the cell, and the rate at which the mRNA is converted into newly synthesized protein (translation). These important pathways are described in detail in Chapter 4. Control of Protein Degradation The life span of intracellular proteins varies from as short as a few minutes for mitotic cyclins, which help regulate passagethrough the mitotic stage of cell division, to as long as the age of an organism for proteins in the lens of the eye. Protein life span is controlled primarily by regulatedprotein degradation. There are two especiallyimportant rolesfor protein degradation. First, degradation removes proteins that are potentially toxic, improperly folded or assembled,or damagedincluding the products of mutated genes and proteins damaged by chemically active cell metabolites. Despite the existenceof chaperone-mediatedprotein folding, it is estimated that as many as 30 percent of newly made proteins are rapidly degraded becausethey are misfolded, their assembly into complexes is defective, or they are otherwise unsuitable. Most other proteins are degraded more slowlS about'l,J percent degradationper hour in mammalian cells. Second,the controlled destruction of otherwise normal oroteins provides a powerful mechanism for maintaining the appropriate levelsofthe proteins and their activities,and for permitting rapid changes in these levels to help the cells respond to changing conditions. Eukaryotic cells have several pathways for degrading proteins. One major pathway is degradation by enzymes within lysosomes, membrane-limited organelles whose acidic interior (pH =4.5) is filled with a host of hydrolytic enzymes. Lysosomal degradation is directed primarily toward aged or defective organelles of the cell-a process

llll+ Animation:The Proteasome 3-29 Ubiquitin-and proteasome-mediated < FIGURE (a)Computer-generated thata imagereveals proteolysis. (blue)at a 195 cap with proteasome structure cylindrical hasa Cytosolic proteins of ubiquitin-tagged target protern eachendof a 20Score Proteolysis are of the core(b)Proteins withinthe innerchamber occurs by polyubiquitination degradation for proteasomal targeted (Ub) of a ubiquitin by attachment E1isactivated Enzyme to a Ub molecule (step this transfers then E) and molecule (E3)transfers ligase o in E2(stepZ) Ubiquitin residue cysteine -NH2 of a on E2to theside-chain the boundUb molecule -NH -CUb (step Ub B) Additional in a targetprotein residue lysine protein by repeatlng target to the areadded molecules chain(step4) The steps[-El, forminga polyubiquitin cap, bythe proteasome targetis recognized polyubiquitinylated of the Ubgroups, to driveremoval whichusesATPhydrolysis proteinintothe of the unfolded andtransfer unfolding, the shortpeptide which from in the core, proteolysis chamber (a)fromw (stepE) lPart arelaterreleased fragments - U b - U b - U b n + 1 digestion of W Baumeister courtesy l etal, 1998,Cell92.357; Baumeister

E

E 1 = u b i q u i t i n - a c t i v a t i negn z y m e E 2 = u b i q u i t i n - c o n j u g a t i negn z y m e E 3 = u b i q u i t i nl i g a s e Ub = u[iqui1i6

I

s ti,iii;J;i,' t

ATPrl

)s

There are severaldistinct regulatory cap complexeswith different activities.The 195 cap has 16-18 protein subunits'6 Release Recognition of which can hydrolyze ATP (i.e., they are ATPases)to prouo U b the energy needed to unfold protein substratesand vide uu ,,n -".j0 transferthem into the inner chamber of the proteaselectively uo ub some. Genetic studiesin yeast have shown that cells cannot \ unfotding survive without functional proteasomes,thus demonstrating orrJ their importance. Furthermore, proper proteasomal activity is so important that cells will expend as much as 30 percent \ cleauage of the energy neededto synthesizea protein to degradeit in

ADP
t-/

\

Dir"hurg"

called autophagy (seeFigure 9-2)-and toward extracellular proteins taken up by the cell. Lysosomeswill be discussedat length in later chapters. Here we will focus on cytoplasmic protein degradationby proteasomes.

l s a C o m p l e xM o l e c u l a r TheProteasome MachineUsedto DegradeProteins Proteasomesare very large macromolecularmachinesconsisting of =50 protein subunitsand having a massof 2-2.4 ', 1'06 Da. They have a cylindrical, barrel-like catalytic core called the 20S protedsome(where S is a Svedbergunit basedon the sedimentationproperties of the particle and is proportional to its size).Bound to the ends of this core are one or two cap complexesthat regulate proteasomal activity. There are approximately 30,000 proteasomesin a typical mammalian cell. There are multiple forms of proteasomes.The best studied of theseis the 265 proteasome(Figure3-29a1,which has a catalyticcore approximately14.8 nm tall and 11.3 nm in diameter and a 19S cap regulatory particle at each end.

a Droteasome. The proteasomal catalytic core comprises two inner rings, with six proteolytic active sitesfacing toward the inner chamber of the =1..7-nm-diameterbarrel' and two outer rings that control substrateaccess.Proteasomescan degrade most proteins thoroughly becausethey have active sites(two each) that cleaveafter hydrophobic residues,acidic residues, and basic residues. Polypeptide substrates must enter the chamber via a regulated aperture at the center of the outer rings. In the 265 proteasome,the opening of the aperture, which is narrow and often allows the entry of only unfolded proteins, is controlled by ATPasesin the 195 cap. The short peptide products of proteasomal digestion (2-24 residues long) exit the chamber and are further degradedrapidly by cytosolic peptidases,eventually being converted to individual amino acids. Some have quipped that a proteasomeis a "cellular chamber of doom" in which proteins suffer a "death of a thousandcuts'" Inhibitors of proteasomefunction can be used therapeutically.Becauseof the global importance of proteasomes for cells, continuous, complete inhibition of proteasomeskills cells. However, partial' discontinuous p.ot.u.o-. inhibition has been introduced as an approach io .".r.., chemotherapy.To survive and groq cells normally DEGRADATION l: PROTEIN FUNCTION R E G U L A T I NPGR O T E I N

87

require the robust activity of a regulatory protein called NF^B, as well as other similar "pro-survival" proteins. In turn, NF*B can function fully and promote survival only when its inhibitor, I^B, is disengagedand degradedby proteasomes(Chapter 16). Partial inhibition of proteasomal activity by a small-moleculeinhibitor drug results in increasedlevels of I*B and, consequently,reducedNF^B activity (loss of pro-survival activity). Cells subsequentlydie by a mechanismcalled apoptosis (programmedcell death, Chapter 21). Becauseat leasrsome types of tumor cells are more sensitive to being killed by proteasome inhibitors than normal cells are, controlled administration of proteasome inhibitors (at levelsthat kill the cancer cells but not normal cells) has proved ro be an effectivetherapy for at least one type of lethal cancer,multiple myeloma. I

U b i q u i t i nM a r k sC y t o s o l i cP r o t e i n sf o r Degradationin Proteasomes If proteasomesare to rapidly degradeonly thoseproteins that are either defectiveor scheduledto be removed,they must be able to distinguish between those proteins that need to be degradedfrom most of the proteins that don't. To solve this problem, cells identify proteins that should be degraded by covalently attaching multiple copiesof a76-residuepolypeptide called ubiquitin that is highly conservedfrom yeast to humans. A complex sensingsystemhas evolved to determine which proteins are to be degraded,and then a three-step processis usedto polyubiquitinylatethe targetproteins.The 195 regulatory cap of the 265 proteasomethen recognizes the ubiquitin-labeledproreins, and unfolds and transports them into the proteasomefor degradation. The ubiquitination process(Figure 3-29b) involves: 1. Activation of wbiquitin-actiuating enzyme(E1)by the addition of a ubiquitin molecule,a reactionthar requires ATP 2. Transfer of this ubiquitin molecule to a cysteineresidue in ubiquitin- conjugating enzyme (E2) 3. Formation of an isopeptidebond betweenthe carboxyl terminus of the ubiquitin bound to E2 and the amino group of the side chain of a lysine residuein rhe targer prorein, a reaction catalyzedby ubiquitin-protein ligase (E3). Subsequent ligasereactionscovalently attach additional ubiquitins to the side chain of lysine 48 of the previously added ubiquitin to generatea linear polymer of ubiquitins, or a polyubiquitin-modifiedtargetprotein.

Specificity of Degradation Thrgetingof specificproteinsis primarily achievedthrough the substrate specificity of the E3 ligase.Thereare hundredsof E3 ligasesin mammaliancells,ensuring that the wide variety of proteins to be polyubiquitinylated can be modified when necessary. An example of the control of the activity of a key cellular protein by the ubiquitin-proteasomesystem is the regulated 88

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degradationof proteins called cyclins,which control the cell cycle (Chapter 20). Cyclins contain the internal sequence Arg-X-X-Leu-Gly-X-Ile-Gly-Asp/Asn (X can be any amino acid), which is recognizedby specificubiquitinylating enzyme complexes.At a specifictime in the cell cycle, each cyclin is phosphorylated by a cyclin kinase. This phosphorylation is thought to causea conformational change that exposesthe recognition sequenceto the ubiquitinylating enzymes,leading to polyubiquitination and proteasomaldegradation. Multifunctional Ubiquitin Tagging Someubiquitination performs cell functions other than the degradation of a t^rgetedprotein. Examplesof alternativeubiquitination schemes include (1) the covalentaddition of a singleubiquitin molecule (monoubiquitination) to a lysine on a rarget protein, (2) the addition of multiple single ubiquitins (multiubiquitination), (3) Iinking the ubiquitin to rhe N-terminus of the target protein, and (4) polyubiquitination in which the ubiquitins are linked to one another via their Lys-63residueinsteadof at the Lys-48 position. These modifications can influence the trafficking (sorting) of proteins within a cell (e.g.,internalization from the cell surface),control DNA repair and regulation of transcription,and undoubtedlyperform numerousother functions yet to be discovered.Cells also have a variery of deubiquitinylating enzymes that can remove ubiquitins from the targetproteinsand thus introducethe possibilityin somecases of reversingthe regulationcausedby the initial ubiquitination.

Regulating Protein Function l: Protein Degradation r Proteins may be regulated at the level of protein synthesis,protein degradation,or the intrinsic activity ofproteins through noncovalent or covalent interactions. r The life span of intracellular proteins is largely determined by their susceptibilityto proteolytic degradation. r Many proteins are marked for destruction with a polyubiquitin tag and then degradedwithin proteasomes,large cylindrical complexeswith multiple proreasesin their interiors (seeFigure3-29). r Variations in the nature of the covalent attachment of ubiquitin to proteins are involved in cellular functions other than proteasome-mediateddegradation, such as changesin the location or activity of proteins.

RegulatingProteinFunctionll: f[ Noncovalent and CovalentModifications The intrinsic activities of proteins are modulated by both noncovalent and covalent changesin the protein. Noncovalent modifications usually involve the binding or dissociation of a molecule and a consequentchangein the conformation of the protein. Often, in such cases,protein activation involves the releaseor rearrangementof an inhibitory subunit or domain.

Covalentmodifications include hydrolysisof the polypeptide chain or addition of a molecule to the side chain of one or more residuesor to the N- or C-terminus of the protein. Such modifications can cause a conformational change in the protein that can alter its activity (form is function). Covalent modifications can also modify the shape of a protein without changing the conformation of the polypeptide and its side chains, for example, by adding a charge or bulky group that can alter the ability of the protein to bind to other molecules.Lastly, covalent modifications can direct the protein to particular locations in a cell (e.g.,the cytoplasmic surface of the plasmamembrane). Many noncovalent and covalent modifications are reversible,thus allowing the activity of an individual protein to be enhancedor suppressedmultiple times during the lifetime of the protein. Others,suchas proteolysis,are irreversible and can be supersededonly by degradationof the modified protein and synthesis of a replacement. In the case of enzymes,these regulatory modifications alter K-, V-"*, or both. Nature has devisedmany different strategiesfor noncovalent and covalent regulation of activity. Here we discuss some common mechanisms for regulating protein function; additional examples will be describedin other chapters.

N o n c o v a l e nB t i n d i n gP e r m i t sA l l o s t e r i c , or Cooperative,Regulationof Proteins One of the most important mechanismsfor regulatingprotein function is through allosteric interactions. Broadly speaking,allostery (from the Greek "other shape") refers to any changein a protein's tertiary or quaternary structure, or in both, induced by the noncovalent binding of a ligand. lfhen a ligand binds to one site (A) in a protein and induces a conformational changeand associatedchangein activity of a different site (B), the ligand is called an allosteric effector of the protein, while site A is called an allosteric binding site, and the protein is called an allosteric protein. By definition, allostericproteins have multiple binding sitesfor either a single type of ligand or for multiple different ligands. The allosteric change in activity can be positive or negative' i.e., can induce an increaseor a decreasein protein activity. Allosteric regulation is particularly prevalent in multimeric enzymesand other proteins where conformational changes in one subunit are transmitted to an adiacent subunit. Cooperatiuity is a term often used synonymously with allostery,and usuallyrefersto the influence(positiveor negative) that the binding of a ligand at one site has on the binding of another moleculeof the sametype of ligand at a different site. Hemoglobin presentsa classicexample of positive cooperative binding in that the binding of a singleligand, oxygen, increasesthe affinity of the binding of the next oxygen molecule.Each of the four subunits in hemoglobin contains one heme molecule. The heme groups are the oxygen-binding componentsof hemoglobin(seeFigure3-13).The binding of oxygen to the heme molecule in one of the four hemoglobin subunits inducesa local conformational changewhose effect

c =5U U)

a

0

20 4 It

40 60 80 Poz(torr)

p O 2i n c a P i l l a r i e s of activemuscles

100 t

P O 2i n a l v e o l i of lungs

3-30 Hemoglobinbindsoxygen FIGURE a EXPERIMENTAL proteinhasfour hemoglobin Eachtetrameric cooperatively. allthesitesareloadedwith sites;at saturation oxygen-binding asthe measured iscommonly concentration Theoxygen oxygen. the oxygen(pOz) pO2 half at which is Pso the partialpressure it is areoccupied; concentration bindingsitesat a givenhemoglobin Thelarge reaction. to the K' for an enzymatic analogous somewhat changein theamountof oxygenboundovera smallrangeof pO2 tissues of oxygenin peripheral permits unloading efficient values percent plot saturation of of a shape The sigmoidal muscle suchas bindingIn of cooperative isindicative ligandconcentration versus a bindingcurveisa hyperbola, binding, of cooperative the absence 1995, fromL Stryer, 3-22 [Adapted in Figure to thecurves similar Company.l Freeman and W H ed, Biochemistru,4th

spreadsto the other subunits, lowering the K- (increasing the affinity) for the binding of additional oxygen molecules to the remaining hemes and yielding a sigmoidal oxygenbinding curve (Figure3-30)' Becauseof the sigmoidalshape of the oxygen-saturationcurve, it takes only a fourfold increasein oxygen concentration for the percent saturation of the oxygen binding sitesin hemoglobin to go from 10 to 90 percent- Conversely,if there were no cooperativity and the shapeof the curve was typical of that for Michaelis-Mententype binding, it would take an 81-fold increase in oxygen concentration to accomplish the same increasein loading. This cooperativity permits hemoglobin to take up oxygen very efficiently in the lungs where the oxygen concentration is high, and unload it in tissueswhere the concentration is low. Thus, cooperativity amplifies the sensitivity of a system to concentration changesin its ligands, providing in many casesselectiveevolutionary advantage. Negative cooperativity often involves the end product of a multistep biochemical pathway, which binds to and reducesthe activity of an enzymethat catalyzesan early, ratecontrolling step for that pathway. In this way excessive buildup of the product is prevented.This kind of regulation of a metabolic pathway is also called end-product inhibition or feedback inhibition.

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89

N o n c o v a l e nB t i n d i n go f C a l c i u ma n d G T pA r e Widely UsedAs AllostericSwitchesto Control ProteinActivity

( a ) C a l m o d u l i nw i t h o u t c a l c i u m

Unlike oxygen, which causesgraded allostericchangesin the activity of hemoglobin, other allosteric effectors act as switches,turning the activity of many different proteins on or off. Two important allosteric switches that we will encounter many times throughout this book are Ca2* and GTP. Ca2+/Calmodulin-Mediated Switching The concentration of Ca2* fteein the cytosol (not bounJto moleculesother than water) is kept very low (=10 7 M) by specializedmembrane transport proteins that continually pump Ca2+ out of the^cytosol.However,as we learn in Chapter 11, the cytosolic Ca'- concentrationcan increasefrom 10- to 100-fold when Ca2*-permeablechannelsin the cell surfacemembranesopen and allow extracellularCa2* ro flow into the cell. This risi in cytosolicCa2* is sensedby specializedCa2*-binding proteins, which alter cellular behavior by turning other prorelns on or off. The importanceof extracellular Ca2* for cell activity was first documentedby S. Ringer in 1883, when he discovered that isolated rat hearts suspendedin a NaCl solution made with 'hard' (Ca2*-rich) London rap warer contracted beautifully whereasthey beatpoorly and stoppedquickly if distilled water was used. Many of the Ca2*-bindingproteinsbind Ca2* using the EF hand/helix-loop-helix structural motif discussedearlier (seeFigure3-9b). The prototype EF hand prorein, calmodulin, is found in all eukaryotic cells and may exist as an individual monomenc protein or as a subunit of a multimeric protein. A dumbbell-shapedmolecule, calmodulin contains four Ca'--binding EF hands with K6's of =10-6 M. The binding of Ca2* to calmodulin causesa conformational changethal permits Ca2*/calmodulin to bind to conservedsecuencesin various target proteins, thereby swirching their activities on or off (Figure 3-31). Calmodulin and similar EF hand proteins thus function as sulitch proteins, acting in concert with changes in Ca2* levels to modulate the activity of other proteins. Switching Mediated by Guanine Nucleotide-Binding Proteins Another group of intracellular switch proteins constitutes the GTPase superfamily. As the name suggests,these proteins are enzymes, GTPases, that can hydrolyze GTp (guanosinetriphosphate) to GDp (guanosinediphosphate). They include the monomeric Ras protein (seeFigure 3-8) and the G. subunit of the trimeric G proteins, both discussedat length in Chapter 15. Both Ras and Go can bind to the plasma membrane,function in cell signaling,and play a key role in cell proliferation and differentiation. Other membersof the GTpase superfamily function in protein synthesis,the transport of proteins between the nucleus and the cytoplasm, the formation of coated vesiclesand their fusion with target membranes,and rearrangemenrsof the actin cytoskeleton.The Hsp70 chaperone proteln we encounteredearlier is an example of an ATp/ADp switch, similar in many respectsto a GTp/GDp switch.

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(bl Caz*/calmodulinbound to target peptide

A FIGURE 3-31 Conformational changesinducedby Ca2* bindingto calmodulin.Calmodulin isa widelydistributed cytosolic proteinthatcontains fourCa2*-brnding sites,onein eachof itsEF handsEachEFhandhasa helix-loop-helix motif At cytosolic Ca2+ concentrations aboveabout5 x 10-7M, bindingof Ca2*to calmodulin changes the protein's conformation fromthe dumbbellshaped, unboundform(a)to onein whichhydrophobic sidecharns becomemoreexposed to solvent. Theresulting Ca2*/calmodulin can wraparoundexposed (b),thereby helices of various targetproteins altering theiractivity.

AII the GTPase switch proteins exist in two forms, or conformations(Figure 3-32):(1) an active("on")form with bound GTP that modulates the activity of specific target proteins to which they bind and (2) an inactive ( ,.off ', ) form with bound GDP, which is generatedby the relatively slow hydrolysis of the GTP bound to the active form. The amount of time any given GTPaseswitch remains active depends on the rate of its GTPase activity. Thus the GTpase activity acts as a timer to control this switch. Cells contain a variety of proteins that can modulate the baseline (or intrinsic) rate of GTPase activity for any given GTpase switch. For example, GTPase activity can be enhanced by specific GTPase-activating proteins, called GAPs, or depressedby other proteins acting as allosteric regulators. After the switch has beenturned off (GTP hydrolysis), it can be turned back on by GTP exchange factor (GEF), which replacesthe bound GDP with a different GTp molecule from the surrounding fluid. Thus, cells can control when the switch is turned on and how long the switch remains on. 'We examine the role of various GTPase switch proteins in regulating intracellular signaling and other processesin several later chaoters.

Active ("on")

GAPs nGSs GDls

* <* *

GEFs4+ Inactive("off")

3-32 The GTPaseswitch. Conversion of the active, A FIGURE intothe inactive formby hydrolysis of GTPis GTP-bound GTPase proteins) andRGSs accelerated by GAPs(GTPase-activating (regulators of G proteinsignaling) andinhibited by GDls(guanine inhibitors) Reactivation by replacing GDPwith nucleotide dissociation (guanine factors). by GEFs nucleotide exchange GTPis promoted

P h o s p h o r y l a t i oann d D e p h o s p h o r y l a t i o n CovalentlyRegulateProteinActivity One of the most common mechanismsfor regulating protein activity is phosphorylation, the addition of phosphate groups to hydroxyl groups on serine,threonine, or tyrosine residues. Protein kinases catalyze phosphorylation, and phosphatases catalyze deph osph orylation. The counteracting activities of kinasesand phosphatasesprovide cells with a "switch" that can turn on or turn off the function of various proteins (Figure3-33). Phosphorylationchangesa protein's chargeand generallyleadsto a conformational change; these effects can significantly alter ligand binding or other featuresof the protein, leading to an increaseor decreasein lts actlvlty.

Active

o tl

R-O-P-O-

Hzo Protein phosphatase

P.

R-OH Inactive

3-33 Regulationof protein activity by the A FIGURE and kinase/phosphatase switch.Thecyclicphosphorylation mechanism for of a proteinisa commoncellular dephosphorylation proteinactivityIn thisexample, the targetproteinR is regulating (bottom) when andinactive active(top)whenphosphorylated responses to havetheopposite someproteins dephosphorylated; phosphorylation

Nearly 3 percent of all yeast proteins are protein kinases or phosphatases,indicating the importance of phosphorylation and dephosphorylation reactions even in simple cells. All classes of proteins-including structural proteins, scaffolds, enzymes, membrane channels' and signaling molecules-have members regulated by kinase/phosphatase switches.Different protein kinases and phosphatasesare specific for different target proteins and can thus regulate a variety of cellular pathways, as discussedin later chapters. Some of these enzymesact on one or a few target proteins, whereas others have many targets. The latter are useful in integrating the activities of proteins that are coordinately controlled by a single kinase/phosphatase switch. Frequently,the target of the kinase (and phosphatase)is yet another kinase or phosphatase' creating a cascade effect. There are many examples of such kinase cascades,which permit amplification of a signal and many levels of finetuning control (seeChapter 15).

ProteolyticCleavagelrreversiblyActivatesor lnactivatesSomeProteins Unlike phosphorylation, which is reversible, the activation or inactivation of protein function by proteolytic cleavageis an irreversible mechanism for regulating protein activity. For example,many polypeptidehormones,such as insulin, are synthesizedas long precursors, and prior to secretion from cells some of their peptide bonds must be hydrolyzed for them to fold properly. In some cases'a single long precursor prohormone polypeptidecan be cleavedinto several distinct active hormones. To prevent the pancreatic serine proteasesfrom inappropriately digesting proteins before they reach the small intestines, they are synthesizedas eymogens, inactive precursor proteins. Cleavageof a peptide bond near the N-terminus of trypsinogen (the zymogen of trypsin) by a highly specific protease in the small intestine generatesa new N-terminal residue (Ile-16), whose amino group can form an ionic bond with the carboxylic acid side chain of an internal aspartic acid. This causesa conformation changethat opens the substrate-bindingsite' activating the enzyme. The active trypsin can then activate trypsinogen, chymotrypsinogen, and other zymogens. Similar' but more elaborate,proteasecascades(one proteaseactivating inactive precursorsof others) that can amplify an initial signal play important roles in several systems' such as the blood-clotting cascade.The importance of carefully regulating such systemsis clear-inappropriate clotting could fatally clog the circulatory system, while insufficient clotting could lead to uncontrolledbleeding. An unusual and rare type of proteolytic processing' termed protein self-splicing,takes place in bacteria and some eukaryotes.This processis analogousto editing film: an internal segmentof a polypeptide is removed and the ends of the polypeptide are reioined (ligated). Unlike other forms of proteolytic processing, protein self-splicing is an autocatalytic process,which proceedsby itself without the participation of enzymes.The excisedpeptide appearsto eliminate itself from the protein by a mechanismsimilar to that usedin

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the processingof some RNA molecules (Chapter 8). In vertebrate cells, the processingof some proteins includes selfcleavage,but the subsequentligation step is absent. One such protein is Hedgehog, a membrane-bound signaling molecule that is critical to a number of develoomental (Chapter 15). processes

H i g h e r - O r d eRr e g u l a t i o nI n c l u d e sC o n t r o l of ProteinLocationand Concentration All the regulatory mechanismsheretofore describedaffect a protein locally at its site of action, turning its activity on or off. Normal functioning of a cell, however, also requires the segregationof proteins to particular compartments such as the mitochondria, nucleus, and lysosomes. In regard to enzymes,compartmentationnot only provides an opportunity for controlling the delivery of substrateor the exit of product, but also permits compering reactions to take place simultaneously in different parts of a cell. We describethe mechanismsthat cells use to direct various proteins to different compartmenrs in Chapters 12 and 13.

Protein Regulation ll: Noncovalent and Covalent Modifications r In allostery,the noncovalentbinding of one ligand mol, ecule, the allosteric effector, induces a conformational change that alters a protein's activity or affinity for other ligands. The allosteric effector can be identical in structure to or different from the other ligands, whose binding it affects.The allostericeffector can be a substrate.acuvaror.or inhibitor. r In multimeric proteins, such as hemoglobin, that bind multiple identicalligand molecules(e.g.,oxygen),the binding of one ligand molecule may increase or decreasethe binding affinity for subsequentligand molecules.This type of allosteryis known as cooperativiry. r Several allosteric mechanisms act as switches, turning protein activity on and off in a reversiblefashron. r Two classesof intracellular switch proteins regulatea variety of cellular processes: (1) Ca2+-bindingproteins (e.g., calmodulin) and (2) members of the GTpase superfamily (e.g.,Ras), which cycle between active GTP-bound and inactiveGDP-boundforms (seeFigure 3-32). r The phosphorylation and dephosphorylationofhydroxyl groups on serine,threonine, or tyrosine residueside chains by protein kinases and phosphatasesprovide reversible on/off regulation of numerous proteins. I Many types of covalent and noncovalent regulation are reversible, but some forms of regulation, like proteolytic cleavage,are irreversibre. r Higher-order regulation includes compartmentation of proteins and control of protein concentration.

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fp

Purifying, Detecting, and

Characterizing Proteins A protein often must be purified before its structure and the mechanism of its action can be studied in detail. However, becauseproteins vary in size, charge, and water-solubility, no single method can be used to isolate all proteins. To isolate one particular protein from the estimated 10,000 different proteins in a particular type of cell is a daunting task that requires methods both for separating proteins and for detectingthe presenceof specificproteins. Any molecule,whether protein, carbohydrate,or nucleic acid, can be separated,or resolued,from other moleculeson the basisof their differencesin one or more physical or chemical characteristics.The larger and more numerous the differences between two proteins, the easier and more efficient their separation.The two most widely usedcharacteristicsfor separatingproteins are size,defined as either length or mass, and binding affinity for specific ligands. In this section, we briefly outline several important techniques for separating proteins; these separation techniquesare also useful for the separationof nucleic acids and other biomolecules.(Specialized methods for removing membrane proteins from membranesare describedin Chapter 10 after the unique properties of theseproteins are discussed.)\Wethen consider the use of radioactive compounds for tracking biological activity. FinallS we considerseveraltechniquesfor characterizinga protein's mass,sequence,and three-dimensionalstructure.

CentrifugationCan SeparateParticlesand MoleculesThat Differ in Massor Density The first step in a typical protein purification schemeis centrifugation. The principle behind centrifugation is that two particles in suspension(cells, cell fragments, organelles,or molecules)with different massesor densitieswill settleto the bottom of a tube at different rates. Remember,mass is the weight of a sample (measuredin grams), whereas density is the ratio of its weight to volume (grams/liter).Proreinsvary greatly in mass but not in density.Unlessa protein has an attached lipid or carbohydrate, its density will not vary by more than 15 percentfrom I .37 glcm3,rhe averageprotein density.Heavier or more densemoleculessettle,or sediment. more quickly than lighter or lessdensemolecules. A centrifuge speedssedimentationby subjectingparticles in suspensionto centrifugal forces as great as 1,000,000 times the force of gravity g, which can sedimentparticles as small as 10 kDa. Modern ultracentrifugesachieve these forces by reaching speedsof 150,000 revolutions per minute (rpm) or greater.However, small particles with massesof 5 kDa or lesswill not sedimentuniformly even at such high speeds. Centrifugation is used for two basic purposes: (1) as a preparativetechniqueto separateone type of material from others and (2) as an analytical technique to measurephysical properties (e.9., molecular weight, densiry shape, and equilibrium binding constants) of macromolecules.The sedimentation

(a) Differentialcentrifugation I

Sample is poured into tube

(b) Rate-zonalcentrifugation I

Sample is layered on top of density gradient

Largerparticle Largerparticle

S m a l l e rp a r t i c l e

S m a l l e rp a r t i c l e Low density (low sucrose concentration) Centrifuge Particlessettle accordingto mass

Centrifugal+

Sucrose gradient

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High density ( h i g hs u c r o s e concentration)

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E

Stop centrifuge Collect fractions and do assay

HI

FIGURE EXPERIMENTAL 3-34 Centrifugation techniques separateparticlesthat differ in massor density.(a)In differential a cellhomogenate or othermixtureisspunlong centrifugation, (eg , cellorganelles, cells), the largerparticles enoughto sediment asa pelletat the bottomof thetube(stepE) The whichcollect (e g , soluble proteins, particles acids) remainin the nucleic smaller tube(stepB) whichcanbetransferred to another liquidsupernatant,

constant s of a protein is a measureof its sedimentationrate. The sedimentationconstant is commonly expressedin svedbergs (S), where a typical, large protein complex is about 3-5S, while a eukaryoticribosomeis 80S. The most common initial step Differential Centrifugation purification for tissuesis the separation of protein cells or in water-solubleproteins from insolublecellular material by differential centrifugation. A starting mixture, commonly a cell homogenate(mechanicallybroken cells),is poured into a tube and spun at a rotor speedand for a period of time that forces cell organellessuch as nuclei and large unbroken cells or large cell fragments to collect as a pellet at the bottom; the soluble proteinsremain in the supernatant(Figure3-34a1.The super-

massof Particles Decreasing (b)In rate-zonal a mixtureisspun(stepE) justlong centrifugation, thatdifferin massbut maybe similar molecules enoughto separate into (e.g, globular proterns, RNAmolecules) in shapeanddensity formedby a gradient commonly zoneswithina density drscrete fromthe areremoved Fractions solution. sucrose concentrated to testing(assayed). bottomof the tubeandsubjected

natant fraction then is poured off' and either it or the pellet can be subjectedto other purification methods to separatethe many differentproteinsthat they contain. On the basisof differencesin Rate-Zonal Centrifugation their masses, proteins can be separated by centrifugation through a solution of increasingdensity called a density gradient. A concentratedsucrosesolution is commonly usedto form density gradients.'Whena protein mixture is layeredon top of a sucrosegradient in a tube and subjectedto centrifugation, each protein in the mixture migrates down the tube at a rate conirolled by the factors that affectthe sedimentationconstant.All the proteins staft from a thin zone at the top of the tube and ,.pui"t. into bands, or zones (actually, disks)' of proteins of P U R I F Y I N GD, E T E C T I N GA,N D C H A R A C T E R I Z I NPGR O T E I N S

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{lttt Technique Animation:SDSGel Electrophoresis [

< EXPERIMENTAL FIGURE 3-35 SDS-polyacrylamide gel (SDS-PAGE) electrophoresis separatesproteinsprimarilyon the (a)Initialtreatment basisof their masses. with SDS,a negatively charged detergent, proteins dissociates multimeric anddenatures all (steptr). Duringelectrophoresis, the polypeptide chains the SDSproteincomplexes migrate gel(stepE). throughthe polyacrylamide Smallcomplexes areableto movethroughthe poresfasterthan largeronesThusthe proteins separate intobandsaccording to their proteinbandsarevisualized sizes astheymigrate. Theseparated by staining with a dyeGtepB). (b)Example of SDS-PAGE separation of (detergent allthe proteins in a whole-cell lysate solublized cells): (/eft)the manyseparate proteins, stained appearing almostasa (right)a proteinpurified continuum; fromthe lysateby a singlestep of antibody-affinity chromatography. Theproteins werevisualized by staining with a silver-based dye lPart(b)modified fromB LiuandM Krieger, 2002,J BiolChem277(31):34125-34135 l

I Denature sample with | sodium dodecylsulfate

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Electrophoresis SeparatesMoleculeson the Basisof Their Charge-to-Mass Ratio (b)

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different masses.In this separationtechnique,called rate-zonal centrifugation, samplesare centrifuged just long enough to separatethe moleculesof interest into discretezones (Figure 3-34b). If a sample is centrifuged for too short a time, the different protein moleculeswill not separatesufficiently. If a sample is centrifuged much longer than necessary,all the proteins will end up in a pellet at the bottom of the tube. Although the sedimentation rate is strongly influenced by particle mass, rate-zonal centrifugation is seldom effective in determining precise molecular weights becausevariations in shape also affect sedimentation rate. The exact effects of shape are hard to assess,especially for proteins or other molecules,such as single-strandednucleic acid molecules,that can assumemany complex shapes.Nevertheless, rate-zonal centrifugation has proved to be the most practical method for separatingmany different types of poiy-"r, and particles.A second density-gradienttechnique,called equilibrium density-gradient centrifugation, is used mainly to separateDNA, lipoproteins that carry lipids through the crrculatory system,or organelles(seeFigure 9-26). 94

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Electrophoresisis a technique for separatingmoleculesin a mixture under the influence of an applied elecric field and is one of the most frequently usedtechniquesto study proteins and nucleic acids. Dissolved molecules in an electric field move, or migrate, at a speeddetermined by their charge-tomass (charge:mass)ratio. For example, if two molecules have the same mass and shape,the one with the greater net charge will move faster toward an electrodeof the opposite polarity. SDS-Polyacrylamide Gel Electrophoresis Becausemany proteins or nucleic acidsthat differ in sizeand shapehave nearly identical charge:massratios, electrophoresisof thesemacromoleculesin solution results in little or no separation of molecules of different lengths.However, successfulsepararionof proteins and nucleic acidscan be accomplishedby electrophoresisin various gels (semisolid suspensionsin water similar to the congealedgelatin found in desserts)rather than in a liquid solution. Electrophoretic separation of proteins is most commonly performed in polyacrylamide gels. .il(i'hena mixture of proteins is placedin a gel and an electriccurrent is applied, smallerproteins migrate faster through the gel than do larger proteins because the gel acts as a sieve,with smaller speciesable to maneuver more rapidly through the pores in the gel than larger species. The shapeof a moleculecan also influence its rate of migration (long asymmetricmoleculesmigrate more slowly than spherical onesof the samemass). Gels are cast betweena pair of glassplates by polymerizing a solution of acrylamide monomers into polyacrylamide chains and simultaneously cross-linking the chains into a semisolidmatrix. The pore size of a gel can be varied by adjusting the concentrations of polyacrylamide and the crossIinking reagent.The rate at which a protein moves through a gel is influenced by the gel's pore size and the strength of the electric field. By suitable adjustment of theseparamerers,

proteins of widely varying sizescan be resolved(separated from one another) by polyacrylamide gel electrophoresis (PAGE). In the most powerful technique for resolving protein mixtures, proteins are exposedto the ionic detergentSDS (sodiumdodecylsulfate)beforeand during gel electrophoresis (Figure3-35). SDSdenaturesproteins,in part becauseit binds to hydrophobic side chains, destabilizing the hydrophobic interactions in the core of a protein that contribute to its stableconformation. (SDStreatmentis usually combined with heating in the presenceof reducing agents that break disulfide bonds.) As a consequence,multimeric proteins dissociateinto their subunits,and all polypeptide chains are forced into extended conformations with similar charge:massratios. SDS treatment thus eliminates the effect of differencesin shapein native structures;therefore, chain length, which correspondsto mass, is the principal determinant of the migration rate of proteins in SDSpolyacrylamide electrophoresis(SDS-PAGE).Even chains that differ in molecular weight by lessthan 10 percentcan be resolved by this technique. Moreover, the molecular weight of a protein can be estimatedby comparing the distance that it migratesthrough a gel with the distancesthat proteins of known molecular weight migrate (there is roughly a linear relationship between migration distance and the log of the molecular weight). Proteins within the

(a)

Protein mixture

Separate in first dimension by charge

E

gelscan be extractedfor further analysis(e.g.'identification by methodsdescribedbelow). of Two-Dimensional Gel Electrophoresis Electrophoresis all cellularproteinsby SDS-PAGEcan separateproteinshaving relatively large differencesin mass but cannot readily resolve proteinshaving similar masses(e.g.,a 41-kDa protein versusa 42-kDa protein). To separateproteins of similar masses'another physical characteristicmust be exploited. Most commonlg this characteristicis electric charge' which is determined by the pH and the relative number of the protein's positively and negatively charged groups, which is in turn dependenton the pKu'sof the ionizablegroups(seeChapter2). Two unrelated proteins having similar massesare unlikely to have identical net chargesbecausetheir sequences,and thus the number of acidic and basicresidues,are different. In two-dimensionalelectrophoresis,proteins are separated sequentially,first by their charges and then by their masses(Figure3-36al.In the first step'a cell or tissueextract is fully denatured by high concentrations (8 M) of urea and then layered on a gel strip that contains a continuous pH gradient. SDS cannot be used, becauseits binding changes the charge of the protein. The gradient is formed by ampholytes, a mixture of polyanionic and polycationic molecules, that are cast into the gel' with the most acidic ampholyte at one end and the most basic ampholyte at the

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s lil,i??"?l" by size FIGURE3-36 Two-dimensionalgel A EXPERIMENTAL proteins on the basis of charge and separates electrophoresis mass.(a) In this technique,proteinsarefirst separatedinto bandson focusing(step[) The the basisof their chargesby isoelectric resultinggel strip is appliedto an SD5-polyacrylamide 9el (stepZ), (step B) (b) In this mass and the proteinsare separatedinto spotsby

cells,each fromcultured gelof a proteinextract two-dimensional by canbe detected Polypeptides a singlepolypeptide spotrepresents Each as autoradiography such dyes,ashere,or by othertechniques point(pl)andmolecular by itsisoelectric polypeptide ischaracterized (b)courtesy of J Celis ] weiqht IPart

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opposite end. A charged protein will migrate through the gradient until ir reachesits isoelectricpoint (pI), the pH at which the net charge of the protein is zero. This technique, called isoelectric focusing (IEF), can resolve proteins that differ by only one charge unit. Proteins that have been separated on an IEF gel can then be separatedin a second dimension on the basis of their molecular weights. To accomplish this separation,the IEF gel strip is placed lengthwiseon one outside edge of a sheetlike (two-dimensional, or slab) polyacrylamide gel, this time saturated with SDS. When an electric field is imposed, the proteins will migrate from the IEF gel into the SDS slab gel and then separateaccording to their masses. The sequential resolution of proteins by charge and mass can achieve excellent separation of cellular proteins (Figure 3-35b). For example, two-dimensional gels have been very useful in comparing the proteomes in undifferentiated and differentiated cells or in normal and cancer cells becauseas many as 1000 proteins can be resolvedas individual spots simultaneously.Sophisticatedmethods have been developed to permit the comparison of complex patterns of proteins in two-dimensional gels from related, but distinct,samples(e.g.,tissuefrom a normal versusa mutant individual) to permir identification of differencesin rhe types or amounts of proteins in the samples (seesection on proteomics,below).

Liquid ChromatographyResolvesproteins b y M a s s ,C h a r g e o , r B i n d i n gA f f i n i t y A third common technique for separating mixtures of proteins or fragments of proteins, as well as other molecules,is basedon the principle that moleculesdissolvedin a solution can differentially interact (bind and dissociate)with a particular solid surface, depending on the physical and chemical properties of the molecule and the surface.If the solution is allowed to flow across the surface, then molecules that interact frequently with the surface will spend more time bound to the surface and thus flow past the ,,rrfac. -or. slowly than molecules that interact infrequently with it. In this technique, called liquid chromatography (LC), the sample is placed on rop of a tightly packed column of spherical beadsheld within a glassor plastic cylinder.The samplethen flows down the column, usually driven by gravitational or hydrostatic forces alone or with the assistanceof a pump, and small aliquots of fluid flowing out of the column, called fractions, are collected sequentially for subsequentanalysis for the presenceof the proteins of interest.The nature of the beads in the column determineswhether the separation of proteins dependson differencesin mass, charge, or binding affinity. Gel Filtration Chromatography proteins that differ in masscan be separatedon a column composedof porous beads made from polyacrylamide, dextran (a bacterial polysaccharide), or agarose(a seaweedderivative)-a technique called gel filtration chromatography. Although proteins flow around the spherical beads in gel filtration chromatography they spend 96

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some time within the large depressionsthat cover a bead'ssurface. Becausesmaller proteins can penetrate into thesedepressions more readily than larger proteins can, they travel through a gel filtration column more slowly than larger proteins (Figure 3-37a). (In contrast, proteins migrate throwgh the pores in an electrophoreticgel; thus smaller proteins move faster than larger ones.) The total volume of liquid required to elute (or separateand remove) a protein from a gel filtration column depends on its mass: the smaller the mass, the more time it is trapped on the beads,the greater the elution volume. By use of proteins of known mass as standards to calibrate the column, the elution volume can be usedto estimatethe massof a protein in a mixture. A protein's shapeas well as its masscan influence the elution volume. lon-Exchange Chromatography In ion-exchangechromatographg a secondtype of liquid chromatography proteins are separatedon the basis of differencesin their charges.This techniquemakesuseof speciallymodified beadswhose surfaces are coveredby amino groups or carboxyl groups and thus carry either a positive charge (NHr*) or a negativecharge(COO ) at neutral pH. The proteins in a mixture carry various net charges at any given pH. When a solution of a protein mixture flows through a column of positively charged beads,only proteins with a net negative charge (acidic proteins) adhere to the beads; neutral and positively charged (basic) proteins flow unimpeded through the column (Figure 3-37b). The acidic proteins are then eluted selectivelyfrom the column by passing a solution of increasingconcentrationsof salt (a salt gradient) through the column. At low sah concentratrons, protein moleculesand beads are attracred by their opposite charges. At higher salt concentrations, negative salt ions bind to the positively charged beads, displacing the negatively charged proteins. In a gradient of increasing salt concentration, weakly bound proteins, those with relatively low charge, are eluted first and highly charged proteins are eluted last. Similarly, a negatively charged column can be used to retain and fractionate basic (positively charged) protelns. Affinity Chromatography The ability of proteins to bind specifically to other molecules is the basis of affinity chromatography. In this technique, ligand or orher molecules that bind to the protein of interest are covalently attached to the beads used to form the column. Ligands can be enzyme substrates,inhibitors or their analogues,or other small moleculesthat bind to specificproteins. In a widely usedform of this techniqu e-anti b o dy -aff in ity, or immu n oaffinity, ch r o matography-the attached molecule is an antibody specific for the desiredprotein (Figure 3-37c). (\7e discussantibodies as tools to study proteins next). An affinity column in principle will retain only those proteins that bind the molecule attached to the beads; the remaining proteins, regardlessof their charges or masses, will pass through the column because they do not bind. However, if a retained protein is in turn bound to other molecules, forming a complex, then the entire complex is

(c)Antibody-affi nity chromatography

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proteins through column

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FTGURE 3-37 Threecommonlyusedliquid EXPERIMENTAL techniquesseparateproteinson the basisof chromatographic mass,charge,or affinity for a specificbinding partner.(a)Gel proteins thatdifferin sizeA separates chromatography filtration layered on thetop of a cylinder iscarefully mixture of proteins proteins packed travelthroughthe with porousbeadsSmaller proteins columnmoreslowlythanlargerproteinsThusdifferent in theeluateflowingout of the bottomof thecolumnat emerging in canbe collected elutionvolumes) times(different different chromatography tubes,calledfractions(b)lon-exchange separate packed with proteins thatdifferin netchargein columns separates (shown positive or a here) charge carry either a beads that special havingthe samenetchargeasthe beads charge. Proteins negative proteins having whereas andflow throughthe column, arerepelled

chargebindto the beadsmoreor lesstightly, the opposite Boundproteins-inthiscase, on theirstructures. depending a salt elutedby passing subsequently charged-are negatively As the ions the column (usually of NaClor KCI)through gradient (more bound proteins tightly the displace they bindto the beads, in orderto be released). highersaltconcentration proteins require is of proteins a mixture (c)In antibody-affinity chromatography, with beadsto whicha specific passed througha columnpacked attachedOnlyproteinwith highaffinityfor the iscovalently antibody proteins flow by thecolumn;allthe nonbinding isretained antibody the boundproteiniseluted through.Afterthe columniswashed, the with an acidicsolutionor someothersolutionthat disrupts protein flows out of then released the complexes; antigen-antibody is collected and thecolumn

retained on the column. The proteins bound to the affinity column are then eluted by adding an excessof a soluble form of the ligand or by changing the salt concentration or pH such that the binding to the moleculeon the column is

disrupted. The ability of this technique to separatepartrcular proteins dependson the selectionof appropriate binding partners that bind more tightly to the protein of interest than to other proteins. . P U R I F Y I N GD, E T E C T I N GA,N D C H A R A C T E R I Z I NPGR O T E I N S

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H i g h l y S p e c i f i cE n z y m ea n d A n t i b o d yA s s a y s Can DetectIndividualProteins The purification of a protein, or any other molecule, requires a specific assay that can detect the presenceof the molecule of interest as it is separatedfrom other molecules (e.g.,in column or density-gradientfractions or gel bands or spots).An assaycapitalizeson somehighly distinctivecharacteristicof a protein: the ability to bind a particular ligand, to catalyze a particular reaction, or to be recognized by a specific antibody. An assaymust also be simple and fast to minimize errors and the possibilitythat the protein of interest becomesdenaturedor degradedwhile the assayis performed. The goal of any purification scheme is to isolate sufficient amounts of a given protein for study; thus a useful assaymust also be sensitiveenough that only a small proportion of the available material is consumed by it. Many common protein assaysrequire just 10 e to 1.0 12g of material. Chromogenic and Light-Emitting Enzyme Reactions Many assaysare tailored to detect some functional aspectof a protein.For example,enzymaticactivity assaysare basedon the ability to detect the loss of substrateor the formation of product. Some enzymatic assaysutilize chromogenic substrates, which changecolor in the courseof the reaction. (Somesubstrates are naturally chromogenic; if they are not, they can be linked to a chromogenic molecule.)Becauseof the specificiryof an enzyme for its substrate,only samples that contain the enzyme will change color in the presenceof a chromogenic substrate; the rate of the reaction provides a measure of the quantity of enzymepresent.Enzymesthat catalyzechromogenic reactionscan also be fused or chemically linked to an antibody and used to "report" the presenceor location of an antigen to which the antibody binds (seebelow). Antibody Assays As noted earlier, antibodies have the distinctive characteristicof binding tightly and specificallyto antigens.As a consequence,preparations of antibodies that recognizea protein antigen of interest can be generatedand used to detect the presenceof the protein, either in a complex mixture of other proteins (finding a needle in a haystack, as it were) or in a partially purified preparation of a particular protein. The tight binding of the antibody to its antigen, and thus the presenceof the antigen, can be visualized by labeling the antibody with an enzyme)a fluorescent molecule, or radioactive isotopes. For example, luciferase, an enzyme present in fireflies and some bacteria, can be linked to an anribody. In the presenceof ATp and the substrate luciferin, luciferase catalyzesa light-emitting reaction. In either case,after the antibody binds to the protein of in-

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naturally fluorescent protein found in jellyfish (seeFigure 9-12). Alternatively after the first antibody binds to its target protein, a second, labeled antibody is used to bind to the complex of the first antibody and its target. This combination of two antibodiespermits very high sensitivity in the detection of a targetprotein. To generatethe antibodies, the intact protein or a fragment of the protein is injected into an animal (usually a rabbit, mouse, or goat). Sometimesa short synthetic peptide of 10-15 residuesbasedon the sequenceof the protein is used as the antigen to induce antibody formation. A synthetic peptide, when coupled to a large protein carrier, can induce an animal to produce antibodies that bind to that portion (the epitope) of the full-sized, natural protein. Biosynthetically or chemically attaching the epitope to an unrelatedprotein is called epitope tagging. As we'll see throughout this book, antibodies generatedusing either peptide epitopes or intact proteins are extremely versatilereagentsfor isolating, detecting,and characterizingprotelns. Detecting Proteins in Gels Proteins embedded within a one- or two-dimensionalgel usually are not visible. The two general approachesfor detecting proteins in gels are either to Iabel or stain the proteins while they are still within the gel or to electrophoreticallytransfer the proteins to a membrane made of nitrocellulose or polyvinylidene difluoride and then detect them. Proteinswithin gels are usually stainedwith an organic dye or a silver-basedstain, both detectedwith normal visible light, or with a fluorescent dye that requires specialized detection equipment. Coomassieblue is the most commonly usedorganic dye, typically usedto detect=1000 ng of protein, with a lower limit of detectionof =4-1,0 ng. Silver stainingor fluorescencestainingare more sensitive(lower limit of =1 ng). Coomassie and other stains can also be used to visualize proteins after transfer to membranes;however.the most common method to visualize proteins in these membranes is immunoblotting, commonly called'Westernblotting. Western blotting combines the resolving power of gel electrophoresisand the specificityof antibodies.This multistep procedure is commonly used to separateproteins and then identify a specificprotein of interest. As shown in Figure 3-38, two different antibodies are used in this method, one that is specific for the desiredprotein and a secondthat binds to the first and is linked to an enzymeor other molecule that permits detectionof the first antibody (and thus the protein of interesr to which it binds). Enzymesto which the secondantibody is attachedcan either generatea visible colored product or, by a processcalled chemiluminescence,produce light that can readily be recorded by film or a sensitive detector. The two different antibodies, sometimescalled a "sandwich," are used to amplify the signalsand improve sensitivity.If an antibody is not available,but the geneencodingthe protein is available and can be used to expressthe protein, recombinant DNA methods (Chapter 5) can irrcorporatea small peptide epitope (epitope tagging) into the normal sequenceof the protein that can be detected by a commercially available antibody to that epitope.

Illl+ Technique Animation:lmmunoblotting I

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Chromogenic detection

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Incubatewith enzYmelinkedAb2 (Y); wash excess

Reactwith substrate for Ab2-linkedenzyme

FIGURE 3-38 Westernblotting A EXPERIMENTAL (immunoblotting) combinesseveraltechniquesto resolveand detect a specificprotein. StepE: Aftera proteinmixturehasbeen bands(orspots, throughan SDSgel,theseparated electrophoresed (blotted) fromthe gelonto gel)aretransferred for a two-dimensional removedStepZ: whichit isnot readily a porousmembraneJrom (Abr)specific of antibody isfloodedwith a solution Themembrane for a while.Onlythe protern andallowedto incubate for the desired thisproteinbindsthe antibody, bandcontaining membrane-bound (whoseposition cannotbe molecules forminga layerof antibody point) iswashedto remove Thenthe membrane seenat this

u n b o u nA d b ' . S t e pE : T h e m e m b r a ni sei n c u b a t ewdi t ha s e c o n d andbindsto thefirstAb1 (Ab2)thatspecifically recognizes antibody (e.9., to linked eitheran enzyme iscovalently antibody Thissecond reaction), a chromogenic can catalyze which phosphatase, alkaline can whosepresence or someothersubstance isotope, radioactive and the location Step4: Finally, with greatsensitivity be detected (e.g, by a deep-purple amountof boundAb2aredetected permitting the reaction), fromchromogenic precipitate (and the destred of the mass) therefore mobility electrophoretic (based on band aswellasitsquantity oroteinto be determined,

RadioisotopesAre lndispensableTools l olecules f o r D e t e c t i n gB i o l o g i c aM

Radioisotopes Useful in Biological Research Hundreds of biological compounds (e.g.,amino acids,nucleosides,and numerous metabolic intermediates)labeled with various radioisotopes are commercially available. These preparations vary considerably in their specific actiuity, which is the amount of radioactivity per unit of material, measured in

A sensitivemethod for tracking a protein or other biological molecule is by detecting the radioactivity emitted from radioisotopesintroduced into the molecule. At least one atom in a radiolabeled molecule is present in a radioactive form, called a radioisotope.

ISOTOPE

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dom/mmol) are available.Likewise' commercialpreparations tH-l"b.l.d nucleic acid precursorshave much ligher speoi 1aC-labeled cific activities than those of the corresponding preparations.In most experiments'the former are preferable t".uot. they allow RNA or DNA to be adequatelylabeled alter a shorter time of incorporation or require a smaller cell sample.Various phosphate-containingcompounds in which every phosphorus atom is the radioisotope phosphorus-32 P U R I F Y I N GD, E T E C T I N GA,N D C H A R A C T E R I Z I NPGR O T E I N S

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a^rereadily available. Becauseof their high specific activity, 32P-labeled nucleotides are routinely ur.d to label nucleic acids in cell-freesystems. Labeled compounds in which a radioisotope replaces atoms normally present in the molecule have virtually the same chemical properties as the corresponding nonlabeled compounds. Enzymes,for instance,generally cannot distinguish betweensubstrateslabeledin this way and their nonlabeled substrates.The presenceof such radioactive atoms is indicatedwith the isotopein brackets(no hyphen) as a prefix (e.g., [3H]leucine).In .ontrurt, labeling almost all biomolecules (e.g., protein or nucleic acid) with the radioisotope iodine-125 (12sI)requires the covalent addition of 12sIto a molecule that normally does nor have iodine as part of its structure.Becausethis labelingproceduremodifies the chemical structure, the biological activity of the labeled molecule may differ somewhat from that of the nonlabeledform. The presenceof such radioactive atoms is indicated with the isotope as a prefix with a hyphen (no bracket) (e.g.,125l-trypsin). Standardmethods for labeling proteins with 12sIresult in covalent attachmentof the 125Iprimarily to the aromatic rings of tyrosine side chains (mono- and diiodotyrosine).

disintegrations per minute per small pixel of surface area. These instruments, which can be thought of as a kind of reusable electronic film, are commonly used to quantitate radioactive molecules separated by gel electrophoresisand are replacingphotographic emulsionsfor this purpose. A combination of labeling and biochemical techniques and of visual and quantitative detectionmethods is often employed in labeling experiments.For instance,to identify the major proteins synthesizedby a particular cell type, a sample of the cells is incubated with a radioactive amino acid (e.g., [35S]methionine)for a few minutes, during which time the Lbeled amino acid mixes with the cellular pool of unlabeled amino acids and some of the labeled amino acid is biosynthetically incorporated into newly synthesizedprotein. Subsequently unincorporatedradioactive amino acid is washed away from the cells. The cells are harvested,the mixture of cellular proteins is extractedfrom the cells (for example, by a detergentsolution), and then separatedby gel electrophoresis; and the gel is subjectedto autoradiography or phosphorimager analysis.The radioactive bands correspondto newly synthesizedproteins, which have incorporatei the radiolabeled amino acid. Alternatively, the proteins can be resolved by liquid chromatographS and the radioactivity in the eluted Labeling Experiments and Detection of Radiolabeled fractions can be determinedquantitatively with a counter.To Molecules Whether labeledcompoundsare detectedby audetect only one specificprotein, rather than all the proteins toradiography, a semiquantitativevisual assay or their radioacbiosyntheticallylabeled this way, a specific antibody to the tivity is measuredin an appropriate ',counter," a highly quantiprotein of interestcan be usedto precipitatethe protein away tative assaythat can determine the amount of a radiolabeled from the other proteins in the sample (immunoprecipitation). compound in a sample, depends on the nature of the experiThe precipitate is then solubilized in a detergenr,separating ment. In some experiments,both types of detection are used. the antibody from the protein, and the sampleis subjectedto In one use of autoradiographS a tissue,cell, or cell conSDS-PAGEfollowed by autoradiography.In this type of exstituent is labeledwith a radioactivecompound, unassociated periment, a fluorescent compound that is activated by the radioactivematerial is washed away, and the structure of the radiation is often infusedinto the gel so that the light emitted sample is stabilized either by chemically cross-linking the can be used to detect the presenceof the labeled protein, macromolecules('fixation') or by freezingit. The sample is either using film or a two-dimensional electronicdetector. then overlaid with a photographic emulsion sensitiveto radiPulse-chase experimentsare particularly usefulfor tracing ation. Developmentof the emulsion yields small silver grains changesin the intracellular location of proreins or the modiwhose distribution correspondsto that of the radioactivemafication of a protein or metabolit. ou.i time. In this experiterial and is usuallydetectedby microscopy.Autoradiographic mental protocol, a cell sample is exposed to a radiolabeled studiesof whole cellswere crucial in determiningthe intracelcompound that can be incorporated or otherwiseattachedto lular siteswhere various macromoleculesare synthesizedand a cellular molecule of interest-the "pulse"-for a brief pethe subsequentmovements of these macromoleculeswithin riod of time, then washedwith buffer to remove the unincorporated label, and finally incubated with an unlabeled form of the compound-the "chase" (Figure 3-39). Samplestaken periodically during the chaseperiod are assayedto determine the location or chemicalform of the radiolabel as a function of time. Often, pulse-chaseexperiments,in which the protein Quantitative measurementsof the amount of radioactivis detected by autoradiography after immunoprecipitation ity in a labeledmarerial are performed with severaldifferent and SDS-PAGE,are usedto follow the rate of synthesis,modification, and degradation of proteins by adding radioactive amino acid precursorsduring the pulse and then detectingthe amounts and characteristicsof the radioactiveprotein during the chase.One can thus observepostsyntheticmodifications of the protein that changeits electrophoreticmobility and the rate of degradation of a specificprotein. A classicuse of the pulse-chasetechniquewas in studiesto elucidatethe pathwav traversedby secretedproteins from their site of synihesisin the endoplasmicreticulum to the cell surface(Chapter 14). 100

.

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key component, which provides a measure of the relative abundancesof each of the ions in the sample.The fourth essential component is a computerized data systemthat is used to calibrate the instrument; acquire,store, and processthe resulting data; and often direct the instrument automatically to collect additional specific types of data from the sample' based on the initial observations. This type of automated feedback is used for the tandem MS (MS/MS) peptidesequencingmethods describedbelow. The two most frequently usedmethods of generatingions of proteins and protein fragmentsare (1) matrix-assistedlaser desorption/ionization(MALDI) and (2) electrospray(ES)' In MALbI (Figure 3-40) the peptide or protein sampleis mixed with a low-molecular-weight,W-absorbing organic acid (the matrix) and then dried on a metal tatget. Energy from a laser ionizes and vaporizes the sample producing singly charged molecular ions from the constituentmolecules'In ES (Figure 3-41.a),the sample of peptidesor proteins in solution is converted into a fine mist of tiny droplets by spraying through a

l"j,\'J," ;:::,'J."'Ll3,' "".' il?HT":',i"H experimentscan FIGURE 3-39 Pulse-chase A EXPERTMENTAL track the pathway of protein modificationor movement newlysynthesized within cells.(a)Tofollowthefateof a specific proteinin a cell,cellswereincubated for 0 5 hr with [3sS]methionine proteins, (thepulse) andthe radioactive to labelallnewlysynthesized intothe cellswasthenwashedaway aminoacidnot incorporated (thechase) for varying timesup to Thecellswerefurtherrncubated to weresubjected from each timeof chase 24 hours,andsamples protein(here,the lowto isolate onespecific immunoprecipitation of the immunoprecipitates receptor) SDS-PAGE lipoprotein density permitted visualization of the one followedby autoradiography (p) protein, asa smallprecursor synthesized whichis initially specific (m) of addition form by larger mature modified to a rapidly andthen from proteinwasconverted Abouthalfof the labeled carbohydrates after0.5 hourof p to m duringthe pulse,the restwasconverted to stablefor 6-8 hoursbeforeit begins chaseTheproteinremains (b) (indicated The same intensity). band by reduced be degraded in cellsin whicha mutantformof the wasperformed experiment to converted proteinismadeThemutantp formcannotbe properly thanthe normalprotein the m form,andit is morequicklydegraded 1986, J CellEloi H A Brush, andM Krieger, fromK F Kozarsky, lAdapted 1567-1s7s 102(5) I Roshanl(eab 021-66950639

arated by the mass analyzeron the basis of their mlz. The two most frequently used mass analyzersare timeof-flight (TOF) instruments and ion traps. TOF instruments e*ploit the fact that the time it takes an ion to passthrough the length of the analyzer before reaching the detector is

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MassSpectrometryCan Determinethe Mass and Sequenceof Proteins Mass spectrometry (MS) is a powerful technique for characthe tertzingproteins.MS is particularly useful in determining 'With such inmass of a protein or fragments of a protein. formation in hand, it is also possibleto determinepart of or all the protein's sequence.This method permits the very highly accuratedirect determination of the ratio of the mass (m) of a charged molecule (ion) to its charge (z), ot mlz. Techniquesare then used to deducethe absolutemass of the ion. There are four key features of all mass spectrometers. The first is an ion source,from which charge, usually in the form of protons, is transferred to the peptide or protein molecules.The formation of these ions occurs in the presenceof a high electric field that then directs the chargedmolecular ions into the second key component' the mass analyzer. The mass analyzer,which is always in a high vacuum chamber, physically separatesthe ions on the basis of their differing mass-to-charge(mlz) ratios. The mass-separated ions are subsequentlydirected to strike a detector,the third

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Time 3-40 Molecularmasscan be FIGURE a EXPERIMENTAL laserdesorption/ionization determinedby matrix-assisted massspectrometry'ln a MALDI-TOF time-of-flight (MALDI-TOF) pulses of lightfroma laserionizea proteinor massspectrometer, on a metaltarget(stepE) An that isabsorbed peptidemixture towardthe detector the ionsin the sample fieldaccelerates electric to the isproportional (steosEl andEl).Thetimeto the detector the having (mlz) ions For ratio. mass-to-charge the squarerootof timeto the ionsmovefaster(shorter the smaller samecharge, usingthetimeof flight weightiscalculated Themolecular detector). of a standard PG ROTEINS O AN , D CHARACTERIZIN P U R I F Y I N GD,E T E C T I N G

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m/z EXPERIMENTAL FIGURE 3-41 Molecularmassof proteins and peptidescan be determinedby electrosprayionization ion-trap massspectrometry.(a)Electrospray (ES)ionization proteins converts andpeptides in a solution intohighlycharged gaseous ionsby passing the solution througha needle(forming the droplets) thathasa highvoltageacross it (charging the droplets). Evaporation of thesolvent produces gaseous ionstharenrera mass spectrometer. Theionsareanalyzed by an ion-trapmassanalyzer that thendirectsionsto the detector. (b) Toppanet:Massspectrumof a mixture of threemajorandseveral minorpeptides ispresented asthe relative abundance of the ionsstriking (y axis)asa the detector functionof the mass-to-charge (m/z)ralio(x axis).Bottornpanel:ln an MS/MSinstrument (suchasthe iontrapshownin part(a)),a

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peptideioncanbe selected specific for fragmentatron intosmaller ionsthatarethenanalyzed anddetected. (also TheMS/MS spectrum calledthe product-ion spectrum) provides detailed structural information aboutthe parention,including sequence information for peptidesHere,the ionwith a mlzof 836.47wasselected, fragmented andthe m/z massspectrumof the productionswas measured Notethereis'nolongeran ionwith an m/zoI g36.47 present because it wasfragmented. Fromthevarying sizes of the productions,the understanding that peptidebondsareoftenbroken in suchexperiments, the knownmlzvalues for individual aminoacid fragments, anddatabase information, the sequence of the peptide, FIIVGYVDDTQFVR, canbe deduced. ona fiourefrom [part(a)based part(b),unpublished 5 Carr; datafromS Carr I

proportional to the square root of mlz (smaller ions move fasterthan larger oneswith the samecharge,seeFigure 3-40). In ion-trap analyzers,tunable electric fields are used to cap'trap,' ions with a specific mlz and to sequentially ture, or pass the trapped ions out of the analyzer onto the detector (seeFigure 3-4ta). By varying the electric fields, ions with a wide range of mlz values can be examined one by one, producing a massspectrum,which is a graph of mlz (x axis) versus relative abundance(y axis) (Figure 3-41'b,top panel). In tandem, or MS/MS, instruments, any given parent ion in the original mass spectrum (Figure 3-41b, top panel) can be broken into smallerions by collisionwith an inmass-selected, ert gas, and then the mlz and relative abundancesof the resulting fragment ions measured (Figure 3-41b, bottom panel), all within the samemachinein about 0.1 s per selectedparent ion. This secondround of fragmentationand analysispermits the sequencesof short peptides (<25 amino acids) to be determined, becausecollisional fragmentation occurs primarily at peptide bonds, so the differencesin massesbetweenions correspond to the in-chain massesof the individual amino acids, permitting deductionof the sequencein conjunctionwith databasesequenceinformation (Figure3-41'b,bottom panel). Mass spectrometryis highly sensitive,able to detectas little as 1 x 10-15 mol (100 attomoles)of a peptideor 10 x 10-15 mol (10 femtomoles)of a protein of 200,000 M$(. Errors in mass measurementaccuracyare dependentupon the specific mass analyzer used, but are typically =0.01 percent for peptidesand 0.05-0.1 percent for proteins. As described in the proteomicssectionthat follows, it is possibleto useMS to analyze complex mixtures of proteins as well as purified proteins. Most commonly, protein samples are digested by proteases,and the peptide digestionproducts are subjectedto analysis. An especiallypowerfully application of MS is to take a complex mixture of proteins from a biological specimen, digestit with trypsin or other proteases'partially separate the componentsusing liquid chromatography (LC), and then transferthe solution flowing out of the chromatographic column directly into an ES tandem mass spectrometer.This technique, called LC-MSIMS, permits the nearly continuous analysisof a very complex mixture of proteins' The abundancesof ions determined by mass spectrometry in any given sample are relative, not absolute' values. Therefore, if one wants to use MS to compare the amounts of a particular protein in two different samples(e.g.,from a normal versusa mutant organism), it is necessaryto have an internal standard in the sampleswhose amounts do not differ between the two samples. One then determines the amounts of the protein of interestrelative to that of the standard in each sample.This permits quantitatively accurateintersamplecomparisonsof protein levels.

from the polypeptide and identified by high-pressureliquid chromatography.The polypeptide is left one residueshorter, with a new amino acid at the N-terminus. The cycle is repeated on the ever shortening polypeptide until all the residueshave been identified. Before about 1985' biologistscommonly used the Edman

merous model organismsis expanding rapidly. As discussed in Chapter 5, the sequencesof proteins can be deducedfrom DNA sequencesthat are predictedto encodeproteins' A powerful approach for determining the primary struct,r.. o? an isolated protein combines MS and the use of sequencedatabases.First' the peptide "mass fingerprint" of the peptide mass fingerprint is the irot.i.r is obtained by MS. A of peptides that are generated weights molecular the iist of

ProteinConformationls Determined by SophisticatedPhysicalMethods In this chapter,we have emphasizedthat protein function is dependent on protein structure. Thus, to figure out how a prot.i.t works, its three-dimensional structure must be Lno*n. Determining a protein's conformation requiressophisticated physical methods and complex analysesof the data. \(e briefly describethree methods used .*p.ri-..tt"l to generatethree-dimensionalmodels of proteins' The use of x-ray crystallographyto X-ray €rystallography determine the three-dimensionalstructuresof proteins was pio-

atoms of the crystal scatterthe x-rays' which produce a drfftaction pattern of discretespots when they are interceptedby photographic film or an electronicdetector (Figure 3-42)' Suchpatextremely complex---composedof as many as 25,000 t...r, "..

ProteinPrimaryStructureCan Be Determinedby ChemicalMethods and from Gene Sequences The classicmethod for determining the amino acid sequence of a protein is Edman degradation. In this procedure, the free amino group of the N-terminal amino acid of a polypeptide is labeled, and the labeled amino acid is then cleaved

three-dimensionalelectron density map in hand' one then "fits" a molecular model of the protein to match the electron density, P U R I F Y I N GD, E T E C T I N GA,N D C H A R A C T E R I Z I NPGR O T E I N s

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proteins. To date, the detailed three-dimensionalstructures of more than 10,000 proteins have beenestablished. Cryoelectron Microscopy Although someproteinsreadily crystallize, obtaining crystals of others-particularly large multisubunit proteins and membrane-associatedproteinsrequires a time-consuming trial-and-error effort to find just the right conditions,if they can be found at all. (Growing crystals suitablefor structural studiesis as much an art as a science.) There are several ways to determine the structures of such difficult-to-crystallize proteins. One is cryoelectron microscopy. In this technique, a protein sample is rapidly frozen in liquid helium to preserveits structure and then examined in the frozen, hydrated state in a cryoelectron microscope. pictures of the protein are taken at various anglesand recorded on film using a low dose of electronsto prevent radiation-induced damageto the structure. Sophisticatedcomputer programs analyze the imagesand reconstruct the protein's structure in three dimensions. Recent advancesin cryoelectron microscopy permit researchersto generatemolecular models that can help provide insight into how the protein functions. The use of cryoelectron microscopy and other types of electron microscopy for visualizing cell structures is discussedin Chapter 9.

a proteincan be determined.(a)Basic components of an x_ray crystallographic determination. Whena narrowbeamof x_rays strikes a crystal,partof it passes straightthroughandthe restrsscattered (diffracted) in various directions Theintensity of thediffracted waves, whichformperiodic arrangements of diffraction spots,is recorded on an x-rayfilm or with a solid-state electronic detector. (b)X-raydiffractionpatternfor a proteincrystalcollected on a solid_ statedetector. Fromcomplex analyses of patterns of spotslikethis one,the location of the atomsin a proteincanbe determined. [part

(a)adaptedfrom L Stryer,1995,Biochemistry, 4Ihed, W H Freeman and p 64; part(b)courtesy Company, of J Berger.j

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NMR Spectroscopy The three-dimensionalstrucures of small proteins containing as many as 200 amino acidscan be studied with nuclear magneric resonance (NMR) spectroscopy. In this technique, a concentratedprotein solution is placed in a magnetic field, and the effects of different radio frequencieson the nuclear spin statesof different atoms are measured.The spin state of any atom is influenced by neighboring atoms in adjacent residues,with closely spaced residues having a greater influence than distant residues. From the magnitude of the effect, the distances between residues can be calculated by a triangulation-like process; these distances are then used to generate a model of the three-dimensionalstructure of the protein. Although NMR does not requiie the crystallizarion of a protein, a definite advantage, this technique is limited to proteins smaller than about 20 kDa. However. NMR analvsis can also be applied to isolated protein domains, which can often be obtained as stable structures and tend to be small enough for this technique.

Purifying, Detecting, and Characterizingproteins r Proteins can be separated from other cell components and from one another on the basis of differencesin their physicaland chemicalproperties. r Various assaysare used to detect and quantify Droteins. Some assaysuse a light-producing reactironto generatea readily detectedsignal. Other assaysproduce an amplified colored signal with enzymesand chromogenic substrates. r Centrifugation separatesproteins on the basis of their rates of sedimentation, which are influenced by their massesand shapes.

proteinson the basisoftheir rates separates r Electrophoresis of movement in an applied electric field. SDS-polyacrylamide gel electrophoresis(PAGE) can resolve polypeptide chains differing in molecular weight by 10 percentor less(seeFigure 3-35). Two-dimensional gel electrophoresisprovides additional resolution by separating proteins first by charge (first dimension)and then by mass(seconddimension). r Liquid chromatography separatesproteins on the basis of their rates of movement through a column packed with spherical beads.Proteins differing in mass are resolved on gel filtration columns; those differing in charge, on ionexchange columns; and those differing in ligand-binding properties, on affinity columns, including antibody-based affinity chromatography(seeFigure 3-37).

investigation.This multipronged approach provided deep insight into the function and mechanismsof action of individual interacting proteins' proteins -Ho*.u.r,or relatively small numbers of proteins does studying this one-by-oneapproach to of what is picture global not efficiently provide insights into a organism' entire or tissue, happening in the proteome of a cell,

Proteomicsls the Study of All or a LargeSubset of Proteinsin a BiologicalSYstem The advent of genomics (sequencingof genomic DNA and its associatedtechnologies' such as simultaneous analysis of the levels of all mRNAs in cells and tissues)clearly showed that a

r Antibodies are powerful reagentsused to detect, quantify, and isolate proteins. 'Western blotting, is a frer Immunoblotting, also called quently usedmethod to study specificproteins that exploits the high specificity and sensitivity of protein detection by antibodies and the high-resolution separation of proteins by SDS-PAGE. r Radioisotopesplay a key role in the study of proteins and other biomolecules.They can be incorporated into molecules without changing the chemical composition of the molecule,or as add-on tags.They can be usedto help detect the synthesis,location, processing,and stability of proteins. r Autoradiography is a semiquantitativetechnique for detecting radioactively labeled molecules in cells, tissues,or electrophoreticgels. r Pulse-chaselabeling can determine the intracellular fate of proteinsand other metabolites(seeFigure 3-39). r Mass spectrometry is a very sensitiveand highly precise method of detecting, identifying, and characterizing proteins and peptides. r Three-dimensionalstructuresof proteins are obtained by x-ray crystallography,cryoelectron microscopS and NMR spectroscopy.X-ray crystallography provides the most detailedstructuresbut requiresprotein crystallization. Cryoelectron microscopy is most useful for large protein complexes, which are difficult to crystallize. Only relatively small proteins are amenableto NMR analysis.

El

Proteomics

For most of the twentieth century,the study of proteins was restrictedprimarily to the analysisof individual proteins. For example,one would study an enzymeby determiningits enzyproducts.rate of reaction,requirematic activity (substrates, ment for cofactors,pH, etc.),its structure' and its mechanism of action. In some cases,the relationshipsberweena few enzymesthat participate in a metabolic pathway might also be studied.On a broader scale,the localizationand activity of an enzymewould be examined in the context of a cell or tissue. The effectsof mutations, diseases,or drugs on the expression and activity of the enzyme might also be the subject of

studies: In a given sample (whole organism, tissue,cell' subcellur compartment), what fraction of the whole proteome is expressed(i.e.,which proteinsare present)? r Of those proteins presentin the sample,what are their relative abundances? r What are the relative amounts of the different splice forms and chemically modified forms (e.g.,phosphorylated, methylated, fatty acylated) of the proteins? comr Which proteins are presentin large multiprotein 'lfhat are plexes,anl which proteins are in each complex? interact? the functions of thesecomplexesand how do they r 'When the state (e.g.'growth rate' stageof cell cycle, differentiation, stresslevel) of a cell changes'do the proteins in the cell or secretedfrom the cell changein a characteristic

r Can such fingerprint-like changesbe used for diagnostic purposes?For example, do certain cancersor heart disease iurrr. .hur".teristic changesin blood proteins? Can the proteomic fingerprint help determine if a given cancer is re,irr".t, or sensitiveto a particular chemotherapeuticdrug? Proteomic fingerprints can also be the starting point for studiesof the meihanisms underlying the changeof state' Proteins (and other biomolecules)that show changesthat are diagnostic of a particular state are calledbiomarkers' Can changesin the proteome help define targetsfor drugs suggestmechanismsby which that drug might induce *ic side effects?If so, it might be possibleto engineer modified versionsof the drug with fewer side effects' o PROTEOMTCS

105

Theseare just a few of the questionsthat can be addressedusing proteomics.The methods used to answer thesequestions are as diverseas the questionsthemselves,and their numbers are growing rapidly.

Figure 3-43 outlines the general LC-MS/MS approach in which a complex mixture of proteins is digested with a protease, the myriad resulting peptides are fractionated by LC into multiple, less complex fractions, which are slowly but continuously injected by electrosprayionization into a tandem mass spectrometer.The fractions are then sequentially subjectedto multiple cyclesof MS/MS until sequences of many of the peptidesare determinedand used to identify from databasesthe proteins in the original biological sample. An example of the use of LC-MS/MS to identify many of the proteinsin eachorganelleis seenin Figure 3-44. Cellsfrom murine (mouse)liver tissuewere mechanicallybroken to release the organelles, and the organelles were partially separated by density-gradient centrifugarion. The locations of the organelles in the gradient were determined using immunoblotting with antibodies that recognize previously identified, organelle-specificproteins. Fractions from the gradient

AdvancedTechniquesin MassSpectrometry Are Criticalto ProteomicAnalysis Advances in proteomics technologies(e.g., mass specrrometry) are having a profound effect on the types of questionsthat can be practically studied. For many years,rwo-dimensional gel electrophoresishas allowed researchersto separate,displaS and characterizea mixture of proteins (Figure 3-36). The spots on a two-dimensional gel can be excised,the protein fragmentedby proteolysis(e.g.by trypsin digestion),and the fragments identified by MS. An alternative to this twodimensional gel method is high throughput LC-MS/MS.

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centrifugation 3-rt4 Density-gradient FIGURE < EXPERIMENTAL can be usedto identify manyof the proteinsin and LC-MS/MS

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eukaryotic cell, the yeastSaccharomycescereuisiae'Approxtmately 500 complexeshave been identified, with an average of 4.9 distinct proteins per complex' and thesein turn are involved in at least400 complex-to-complexinteractions'Such systematicproteomic studiesare providing new insights into tire organiiation of proteins within cells and how proteins work together to permit cellsto live and function'

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subcellularlevels. r Proteomicsprovides insightsinto the fundamental organizationof proteins within cells,and how this organization is influencedby the state of the cells (e.g.,differentiation into distinct cell types; responseto stress'disease,and drugs)' r A wide variety of methods are used for proteomic analyses,including two-dimensional gel electrophoresis,densitygradient centrifugation, and mass spectroscopy(MALDITOF and LC-MS/MS)' omics has helped begin to identify the proteomesof les ("organelleproteomeprofiling") and the organof individual proteins into multiprotein complexes eract In a complex network to support life and cellular function

ER/Golgi vesicles 220

Early enoosomes 76

Proteomics combined with molecular geneticsmethods are currently being usedto identify all protein complexesin a P E R S P E C T I VF EO S RT H E F U T U R E

O

107

motif 68 motor proteins 85 peptide bond 55 polypeptides 65 primary structure 66 proteasomes54 protern 66 proteome 64

quaternary str]uctvre 72 r ate-zonal centrifugation 93 secondarystructure 65 tertiary structure 67 ubiquitin 88

v^^*go

'Western blotting 98 x-ray crystallogr aphy 10 3

proteomics105

Review the Concepts

built. Becausesubunits of the complex may already be solved by crystallographSa composite,tru.tur. consistingof the x-ray-derived subunit structuresfit to the EM-deriveJ model will be generated.An example of this approach for an elec_ tron-transport "supercomplex" is describedin Chapter 12. Methods for rapid structure determination combined with identificarion of novel substratesand inhibitors will

range. Routine analysisof specimenswith such diversecon_ centrations should dramatically improve the mechanistic and diagnostic value of blood plasma proteomics.

KeyTerms cr helix 65 activation energy 79 active site 80 allostery 89 amyloid frlament 77 autoradiography100 B sheet 66 B turn 66 chaperones75 108

o

C H A P T E R 3I

conformations 63 cooperativity 89 (tomarn /u electrophoresis94 lr nomotogy /Z K- 90 ligand 78 liquid chromatography 9G molecular machine 64 P R O T E I NS T R U C T U RAEN D F U N C T I O N

7, The three-dimensional structure of a protein is deter_ mined by its primary, secondary,and tertiary structures.Define the primary, secondary, and tertiary structures. What are some of the common secondary structures? ril/hat are the forces that hold together the secondary and tertiary structures? 2. Proper folding of proteins is essentialfor biological ac_ tivity. Describethe roles of molecular chaperonesand chap_ eronins in the folding of proteins. 3. Enzymes can catalyzechemical reactions. How do en_ zymes increasethe rate of a reaction? What constitutes the active site of an enzyme?For an enzyme-catalyzedreaction, what are K- and V-""? For enzymeX, the K- for substrrte A is 0.4 mM and for substrate B is 0.01 mM. \Which sub_ strate has a higher affinity for enzyme X? 4. Motor proteins convert energy into a mechanicalforce. Describethe three generalproperties characteristicof motor proterns,

6, The function of proteins can be regulatedin a number of ways. What is cooperativitg and how does it influence pro_ tein function? Describe how protein phosphorylation and proteolytic cleavagecan modulate protein function. 7. A number oftechniques can separateproteins on the ba_ sis of their differencesin mass. Describe the use of two of thesetechniques,cenrrifugation and gel electrophoresis.The blood proteins rransferrin (M\X/ 76 kDa) and lysozyme(MW 15 kDa) can be separated by rate-zonal centrifugation or SDS-polyacrylamidegel electrophoresis.Which oi the two proteins will sediment faster during centrifugation? Which will migrate faster during electrophoresis? 8. Chromatography is an analytical method used to sepa_ rate proteins. Describethe principles for separatingproteins by gel filtration, ion-exchange,and affinity ihromatography. 9, Various methods have beendevelopedfor detectingpro_ teins. Describehow radioisotopesand autoradiography can be used for labeling and detectingproteins. Ho* doe, Ifr.rt_ ern blotting detectproteins?

10. Physical methods are often used to determine protein conformation. Describe how x-ray crystallography,cryoelectron microscopy,and NMR spectroscopycan be used to d e t e r m i n et h e s h a p eo f p r o t e i n s . 1L. Mass spectrometryis a powerful tool in proteomics.What are the four key features of a mass spectrometer?Describe briefly how MALDI and two-dimensionalpolyacrylamidegel electrophoresis(2D-PAGE)could be usedto identify a protein expressedin cancercellsbut not in normal healthycells.

mic fractions by differential centrifugation. Two-dimensional gels were run, and the stained gels are shown.below' \fhat do you conclude about the cellular localization of proteins 1-7? Control Nu c l e a r 10

4pH

4

Cytoplasmic pH 10

o

a

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Analyze the Data

a

a

Proteomicsinvolvesthe global analysisof protein expression. In one approach, all the proteins in control cells and treated cells are extracted and subsequentlyseparatedusing two-dimensional gel electrophoresis.TypicallS hundreds or thousandsof protein spots are resolvedand the steady-statelevels of each protein are compared between control and treated cells. In the following example, only a few protein spots are shown for simplicity. Proteins are separated in the first dimension on the basis of charge by isoelectricfocusing (pH a-10) and then separatedby sizeby SDS-polyacrylamide gel electrophoresis.Proteinsare detectedwith a stain such as Coomassieblue and assignednumbers for identification. a. Cellsare treatedwith a drug ("+ Drug") or left untreated ("Control"), and then proteins are extractedand separated by two-dimensional gel electrophoresis.The stained gels are shown below. What do you concludeabout the effect of the drug on the steady-statelevelsof proteins 1-7?

12 O.

+ Drug Nuclear pH 10

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Cytoplasmic pH 10

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o

a o o

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d. Summarize the overall properties of proteins 1-7, combiningthe data from parts (a), (b), and (c)' Describehow you could determinethe identity of any one of the proteins'

References General References Berg,J. M., J. L. Tymoczko, and L. Stryer.2007' Biochemistry, 6th ed. W. H. Freemanand ComPanY. Nelson, D. L., and M. M. Cox. 2005. LehningerPrinciplesof Biochemistry,4thed. !(/. H. Freemanand Company'

+ Drug 4pH10

Control 4pH10

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a

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^o t aa

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b. You suspectthat the drug may be inducinga protein kinaseand so repeatthe experimentin part (a) in the pres32P-labeled inorganicphosphate.In this experiment enceof the two-dimensional gels are exposedto x-ray film to detect 32P-labeledproteins. The x-ray films are the presenceof shown below. \fhat do you conclude from this experiment about the effect of the drug on proteins 1-7? Control pH

4

10 a

4

+ Drug pH

10

a

a c. To determinethe cellular localizationof proteins 1-7, the cellsfrom part (a) were separatedinto nuclearand cytoplas-

Entrv site into proterns,structures'genomes'and taxonomy: http://www.ncbi.nlm.nih.govlEntrezl The protein 3D structuredatabase:http://www'rcsb'org/ Structuralclassificationsof proteins: http://scop'berkeley'edu/ Sitescontaininggeneralinformation about protetns: httpy'/www.exp"tyiti; http://www.proweb'org/; http://scop'berkeley' edu/intro.html PROSITEDatabaseof protein families and domarns: te/ http://www.expasy.org/Prosr Domain organizationof proteins and large collection of multiarelPfanl ple sequencealignments:http://www.sanger'ac'uk/Softw Hierarchical Structure of Proteins and Protein Folding Branden,C., and J. Tooze. 1999. Introduction to Protein Strwcture. Garland. BrodsknJ. L., and G. Chiosis.2006'Hsp7Omolecularchaper,oies in human diseaseand identificationof small on.r' .-.tji"g molecule-lod"ulrrotr. Curr. Top. Med- Chem' 6(11'\:1215-1225' Bukau, B, J. Veissman, and A. Horwich' 2006 Molecular chaperonesand ptot.i.t quality control. Cell 125(3):443451" /' Cohen,F.E. 1999.Proteinmisfoldingand prion diseases' Mol. B iol. 293:313-320. Coulson,A. F., and J. Moult. 2002' A unifold, mesofold' and ' superfoldmodel of protein fold use.Proteins46:61'-7"1 mechaunifying a there Daggett,V, and A' R. Fersht.2003' Is ' 28(\l:18-25 Sci' Biocbem' Trends folding? foi-protein nism R E F E R E N C E S.

10 9

Dobson, C. M. 1999. Protein misfolding, evolution, and disease. TrendsBiochem. Sci.24:329-332. Gimona, M.2006. Protein linguistics-a grammar for modular protein assembly?Nat. Reu.Mol. Ceil Biol.7(Il:69_73. Gough,J. 2006. Genomic scalesub-family assignmentof pro_ tein domains.Nucl. Acids Res.34(13):3625-3633. Koonin, E. V., Y. I. Wolf, and G. p. Karev.2002. The srructure of the protein universeand genomeevolution. Nature 420:21,g_223. Lesk, A. M. 2001. Introduction to protein Architecture.Oxford. and H. S. Rye. 2005. GroEl-mediated protein folding: ,\i", V, making the impossible,possible.Crit. Reu.Biochem.Mol. Biol. 4l(4):21.1-239. Orengo, C. A., D. T. Jones,and J. M. Thornton. 1994. protein superfamiliesand domain superfoldi. Nature 372: 631_634. Patthn L. 1999. Protein Euolution. Blackwell Science. . Rochet,J.-C., and P.T. T andsbury.2000. Amyloid fibrillogene_ sis:themesand variations. Curr. Opin. Struc. Bioi. l}:60_6g. . . Voge!,C, and C. Chothia. 2006. prorein family expansionsand biological complexity. PLoS Comput. Biol. 2(5.;e4g. Yaffe,M. 8.2006. "Bits" and pieces.SclSTKE. Jun 20 (3401:pe2g. Y,oung,J. C., et aL.2004.pathways of chaperone-mediated pro_ tein folding in the cytosol. Nat. Reu.Mol. Cet[ glol. 5:7g1._797. Protein Function ^ Dressler,D. H., and H. Potter. 1997. DiscoueringEnzymes. Scientific American Libra ry. Fersht,A. 1999. Enzyme Structureand Mechanism,3d ed. '!7. H. Freemanand Company. Jeffery,C. J.2004. Molecular mechanismsfor multitasking: recentcrystal structuresof moonlighting proteins. Cwrr.Opin."Struc. Biol. l4(61:663-668. Marnett, A. B., and C. S. Craik. 2005. papa'sgot a brand new tag: advancesin identificarionof proteasesani the]. substrates. TrendsB iotechnol. 23 2\: 59-64. L. 2005. The catalytic triad of serinepeptidases.Cell _Polgar, Mol. Life Sci.62(1.9-20\:2t 6I-217 2. Radisky,E. S., et aL.2006.Insightsinto the serineprotease mechanismfrom atomic resolutionltructures of trypsin reacrion inrermediates. Proc. Nat'l Acad. Sci.USA 103(1g);6g35_6g40. Schenone,M, B. C. Furie, and B. Furie. 2004. The blood coagulationcascade.Curr. Opin. H ematol. ll(4\ :272_277. Schramm,V. L. 2005. Enzymatictransition staresand transition stateanalogues. Cun Opin. Struc.Biol. 15(6):604_613. Regulating Protein Function l: protein Degradation Glickman, M. H., and A. Ciechanover.2002Theubicuitin_ proteasomeproteolytic pathway: destructionfor the sakeof construction.Physiol. Reu. 82(2\:373-42g. Goldberg_, A.L.2003. Protein degradationand protection againstmisfolded or damagedproteinl. Nature 426:.g95_g99. Goldberg,.{. L, S.J. Elledge,and J. If. Harper. 2001. The cellu_ , r a rc h a m b e o r t d o o m . S c i .A m . 2 8 4 1 1 1 : O g _ 7 3 . Groll, M., and R. Huber. 2005. purification, crystallization,and ]^rff^f1a!fsi9 of the yeast20S proteasome.Met'h. Enzymol. 398:329-336. Kisselev,A. F., A. Callard, and A. L. Goldberg.2006. Impor_ tanceof rhe.differentproteolytic sitesof the prot;some and the efficacy ot rnhrbrrorsvarieswith the protein subsfrate. Biol. Cbem. J. 281(13):8582-8590. Rechsteiner, M., and C. p. Hilt. 2005. Mobilizing the proteolytic machine:cell biologicalrolesof proreasome acrivatorsand in_ hibitors.TrendsCell Biol. l5(l);27-J3. Zhou. P.2006. REGgamma:a shorrcutto destruction.Cel/ r24(2\:256-257. Zolk, O., C. Schenke, and A. Sarikas .2006.The ubiquitin_ proteasomesysrem:focuson the heart.Cardiouasc.Res.7Oi3\:410_12t.

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Regulating Protein Function ll: Noncovalent and Covalent Modifications Bellelli,A., et al. 2005. The allostericpropertiesof hemoglobin: insightsfrom narural and site directed-uiants. Cwrr.prot. pZo. Sci. 7(t):1745. Burack, \7. R., and A. S. Shaw-2000. Signaltransducrion:hang_ ing on a scaffold.Curr. Opin. Cell Biol. 12:271-216. _ Halling, D. B, P. Aracena-parks,and S. L. Hamilton. 2006. Reg_ uljrtion of voltage-gatedCaz+ channelsby calmodulin. Sci STKE lXn 1.7(3181:er1.. Horovitz, A., et al. 2001. Review: allosteryin chaperonins. /. Struc. Biol. 135:104-1,14. Kern, D., and E. R. Zuiderweg. 2003. The role of dynamicsin allostericregulation.Curr. Opin. Struc. Biol. 13(6):748-757. I..ane,K. T., and L. S. Beese.2006. Thematicreview series:lipid post_ translationalmodifications.Structuralbiology of protein farnesyitrans_ feraseand geranylgeranyltransferase rypeL j. Lip;d Res.4Z(41:tg1,-691. Lim, W. A.2002. The modular logic of signalingproteins:build_ ing allostericswitchesfrom simple binding domains. Curr. Opin. Struc.Biol. 12:61,-68. Martin, C., Y. Zhang.2005. The diversefunctions of histone lysinemethylation.Na/. Reu.Mol. Cell Biol.6(11):838-849. Sawyer,T. K., et al. 2005. Proreinphosphorylationand signal transductionmodulation: chemistryperspeitivesfor small-mo'iecule drug discovery.Med. Chem. l(3)2%-3I9. Xia,Z., and D. R. Storm.2005. The role of calmodulinas a sisnal integrator for synapticplasticity. Nat. Reu.Neurosci. 6(41:267-27 d. Yap, K. L., et al. 1999. Diversiry of conformational statesand changeswithin the EF-hand protein superfarnlly.proteins 37:499_507. Purifying, Detecting, and Characterizing proteins Domon, B., and R. Aebersold.2006. Mass spectrometryand protein analysis.Science3t2(5771):212-217. . Fncarnacion,S., et al. 2005. Comparativeproteomicsusing 2_D gel electrophoresisand massspectrometryas tools to dissectstimu_ lons and regulonsin bacteriawith sequencedor partially sequenced genomes.Biol. Proc. Online 7:ll 7-135. Hames, B. D. A Practical Approach. Oxiord Universiry press.A methods seriesthat describesprotein purification methods and assays. Patton, W. F.2002. Detection,technologies in proteome analysis. J. Chromatogr.B. Analyt. Technol.Biomed. Life SZl.ZZtlt.Zy:l_Zt. White, I. R., et al. 2004. A staristicalcomparisonof silver and !IP\O Ruby staining for proreomic analysis.Electrophoresis 25(17):3048-3054. Proteomics Foster,L. J., et al. 200 . A mammalian organellemap by pro_ tein correlation profiling. Cell 125(1\:187-195. Fu, Q., and J. E. Van Eyk.2006. proteomicsand heart disease: identifying biomarkers of clinical vtj\ty. Expert Reu. proteomics 3(2):237-249. _ Gavin. A. C., et a|.2006. Proreomesurveyrevealsmodularity of the yeastcell machinery.Nature 440(7084):631-636. Kislinger,T., et al. 2006. Global surveyof organ and organelle protern expre_ssion in mouse:combinedproteomicand trans&iptomic profiling. Cell 125(1):173-186. Kolker, E, R. Higdon, and J. M. Hogan. 2006. protein identifi_ cation.andexpressionanalysisusing mass spectrometry.Trends Mi crobi ol. I4\5 l:229-235 . Krogan, N. J., et al. 2006. Global landscapeof protein complexes in the yeastSaccb aromycescereuisiae. Nature-440(i0g4):637_623. Ong, S. E., and M. Mann. 2005. Mass spectrometry_based pro_ teomrcsturns quanritarive.Nar. Chem. Biol. l(5):252_262. Rifai, N., M. A. Gillette, and S. A. Carr.2006. protein bio_ T."lk.f dis.coveryand validation: the long and uncertainparh ro clinical utility. Nat. Biotecb. 24(8\971-t$.

C H A P T ER

BASICMOLECULAR GENETIC MECHANISMS Colored transmissionelectron micrograph of one ribosomal RNA transcription unit from a Xenopus oocyte. Transcription proceeds from left to right,with nascentribosomalribonucleoprotein RNA (rRNPs) growingin lengthas eachsuccessive complexes polymerase I moleculemovesalongthe DNAtemplateat the center eachrRNPis orientedeitheraboveor belowthe ln thisoreoaration so that the overallshapeis centralstrandof DNAbeingtranscribed, rRNPs of a livingcell,the nascent similarto a feather.In the nucleolus L MillerAcience Oscar likea bottlebrushlProfessor extendin alldirections, PhotoLibrary l

he extraordinary versatility of proteins as molecular machines and switches, cellular catalysts' and components of cellular structureswas describedin Chapter 3. In this chapter we consider how proteins are made' as well as other cellular processesthat are critical for the survival of an organism and its descendents.Our focus will be on the vital molecules known as nucleic acids, and how they ultimately are responsible for governing all cellular function. As introduced in Chapter 2, nucleic acids are linear polymers of four types of nucleotides(seeFigures 2-13' 2-1'6 and 2-1,7).These macromolecules( 1 ) contain in the precise sequenceof their nucleotidesthe information for determining the amino acid sequenceand hence the structure and function of all the proteins of a cell, \2) are critical functional components of the cellular macromolecular factories that select and align amino acids in the correct order as a polypeptide chain is being synthesized,and (3) catalyze a number of fundamental chemical reactions in cells, including formation of peptide bonds between amino acids during protein synthesis. Deoxyribonucleicacid (DNA)is an informational molecule that contains in the sequenceof its nucleotidesthe information required to build all the proteins of an organism, and hencethe cells and tissuesof that organism. It is ideally suited to perform this function on a molecular level. Chemically, it is extraordinarily stable under most terrestrial conditions, as exemplified by the ability to

stored in the sequenceof the four possible nucleotides that

OUTLINE 4.',|

Structureof NucleicAcids

113

4.2

Transcriptionof Protein-CodingGenes and Formationof FunctionalmRNA

120

4.3

The Decodingof mRNA bY tRNAs

127

4.4

StepwiseSynthesisof Proteinson Ribosomes 132

4.5

DNA Replication

139

4.6

DNA Repairand Recombination

145

4.7

of the Cellular Viruses:Parasites GeneticSYstem

111

make up the -3 x 10e base pairs in the human genome. Becauseof the principles of basepairing discussedin this chapter, the information is readily copied with an error rate of <1 in 10e nucleotides per geniration. The exact replication of this information in any speciesassuresits genetic continuity from generation to generation and is critical to the normal developmenrof an individual. DNA fulfills these functions so well that it is the vessel for genetic information in all forms of life known. (One exception is RNA viruses; however, these are limited to extremely short genomesbecauseof the relative instability of RNA compared with DNA, as we will see.)The discovery that virtually all forms of life use DNA to encodetheir genetic information, and also use nearly the same nucleic acid sequencecode to specifyamino acid sequence,implies that all forms of life descendedfrom a common ancesror on the basis of storageof information in nucleic acid sequence.This information is accessedand replicated by specific base pairing between bases. The information stored in DNA is arrangedin hereditary units, now known as genes,which control identifiable traits of an organism. In the processof transcription, the information stored in DNA is copied into ribonucleic acid (RNA), which has three distinct roles in protein synthesis. Portions of the DNA nucleotide sequenceare copied into

lation becausethe nucleotide sequencelanguage of DNA and RNA is translated into the amino acid sequencelan_ guageof proteins. Discovery of the structure of DNA in 1953 and subse_ quent elucidation of how DNA directs synthesisof RNA, which then directs assemblyof proteins-the so-called,cen_

decoded into a variety of proteins in the correct cells at the correct times in development. This regulation of gene expressionallows hemoglobin to be expressedonly in cells of the bone marrow (reticulocytes)destinedto develop into

1',t2

CHAPTER 4

I

circulating red blood cells (erythrocytes),and directs developing neurons to make the proper synapses(connections) with 1011other developingneurons in the human brain. The fundamental molecular generic processesof DNA replication, transcription, and translation must be carried out with extraordinary fidelity, speed,and accurate regulation in or, der for organismsas complex as prokaryotes and eukaryotes to develop normally. This is achievedby chemical processes that operate with extraordinary accuracycoupled with multiple layers of checkpoint, or surveillance,mechanismsthat test whether critical steps in these processeshave occurred correctly before the next step is initiated. The highly regulated expressionof genesnecessaryfor the developmento1 a multicellular organism requires integrating information from signalssent by distant cellsin the developingorganism, as well as from neighboring cells, and an intrinsic developmental program determined by earlier stepsin embryogenesis taken by that cell's progenitors. All this regulation is dependent on control sequencesin the DNA that function with proteins called transcription factors to coordinate the expressionof every gene. RNA sequenceswe discussin Chapter 8 that regulateRNA processingand translation also are encoded in DNA originally. Thus nucleic acids function as the "brains and central nervous system" of the cell, while proteins carry out the functions they specify. In this chapter,we first review the structuresand properties of DNA and RNA, and explore how the different characteristicsof each type of nucleic acid make them suited for their respectiveroles in the cell. In the next severalsections we discussthe basic processessummarizedin Figure 4-1: transcription of DNA into RNA precursors, processing of these precursorsto make functional RNA molecules.translation of mRNAs into proteins, and the replication of DNA. After outlining the individual roles of -RNA, IRNA, and rRNA in protein synthesis,we presenta detailed description of the components and biochemical stepsin translation. .We also consider the molecular problems involved in DNA replication and the complex cellular machinery for ensuring accuratecopying of the geneticmaterial. Along the way, we compare theseprocessesin prokaryotes and eukaryotes.The next section describeshow damageto DNA is repaired, and how regions of different DNA molecules are exchangedin the processof recombination to generatenew combinations of traits in the individual organisms of a species.The final section of the chapter presents basic information about viruses, which, in addition to being significant pathogens, are important model organisms for studying macromolecular synthesisand other cellular processes.Viruses have relatively simple structures compared with cells, and small genomesthat made them tractable for historic early studies of the basic processesof DNA replication, transcription, translation, recombination, and gene expression.Viruses continue to teach important lessonsin molecular cell biology today and have been adapted as experimental tools for introducing any desired genes into cells, tools that are currently being tested for their effectivenessin human gene rnerapy.

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4-1 Overviewof four basicmoleculargenetic A FIGURE that leadto we coverthethreeprocesses processes. In thischapter (n-B) andthe process DNA for replicating production of proteins (4) Because theyhavebeen machinery, viruses utilizehost-cell Duringtranscriptton for studying theseprocesses models important (n), thefour-base DNA geneby RNApolymerase of a protein-coding or of a proteiniscopied, the aminoacidsequence codespecifying by the RNA(pre-mRNA) messenger into a precursor transcribed, (rNTPs) monomers triphosphate polymerization of ribonucleoside to the sequences andothermodifications of noncoding Removal producea (Z), collectively processing, pre-mRNA knownasRIVA During to the cytoplasm whichistransported mRNA, functional

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Structureof NucleicAcids

DNA and RNA are chemically very similar. The primary structuresof both are linear polymerscomposedof monomers called nucleotides.Both function primarily as informational molecules,carrying information in the exact sequenceof their nucleotides.Cellular RNAs range in length from fewer than one hundred to many thousandsof nucleotides.Cellular DNA moleculescan be as long as severalhundredmillion nucleotides.These large DNA units in associationwith proteins can be stained with dyes and visualized in the light microscope as chromosomes,so named becauseof their stainability. Though chemically similar, DNA and RNA exhibit some very important differences.For example, RNA can also function as a catalytic molecule.As we'll see,it is

intothe (B), thefour-base codeof the mRNAisdecoded translation the macromolecular of proteinsRibosomes, acidlanguage 2O-amino of two subunits themRNAcode,arecomposed thattranslate machines (rRNAs) andmultiple RNAs fromribosomal in the nucleolus assembled subunits ribosomal cytoplasm, (/eft). to the Aftertransport proteins the help with protein synthesis out carry and mRNA with an associate factorsDuringDNA (tRNAs) translation andvarious RNAs of transfer to divide, (4), whichoccurs onlyin cellspreparing replication (dNTPs) arepolymerized monomers triphosphate deoxyribonucleoside molecule' DNA chromosomal each of copies identical to yieldtwo copies oneof the identical cellreceives Eachdaughter

the different and unique properties of DNA and RNA that make them each suited for their specificroles in the cell'

A N u c l e i cA c i d S t r a n dl s a L i n e a rP o l y m e r w i t h E n d - t o - E n dD i r e c t i o n a l i t Y In all organisms, DNA and RNA each comprise only four different nucleotides. Recall from Chaptet 2 that all nucleotidesconsist of an organic base linked to a five-carbon sugar that has a phosphate group attached to carbon 5' In RNA, the sugar is ribose; in DNA, deoxyribose(seeFig,r,re2-16). The nucleotides used in synthesisof DNA and RNA contain one of five different bases.The basesadenine (A) and guanine(G) arepurines,whichcontainapaftof fused rings; the basescytosine (C), tbymine (T), and uracil (U) are

ACIDS S T R U C T U ROEF N U C L E I C

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erty of the molecule. The chemical linkage berweenadjacent nucleotides,commonly called a phosphodiesterbond, actually consistsof two phosphoesterbonds, one on the 5, side of the phosphate and another on the 3' side. The linear sequenceof nucleotideslinked by phosphodiesterbonds constitutesthe primary structureof nucleic acids. Like polypeptides, polynucleotides can twist and fold into three-dimensionalconformations stabilized by noncovalent bonds. Although the primary structures of DNA and RNA are generally similar, their three-dimensionalconformations are quite different. Thesestructural differencesare critical to the different functions of the two types of nucleic acids.

N a t i v eD N A l s a D o u b l eH e l i xo f C o m p l e m e n t a r y A n t i p a r a l l eS l trands The modern era of molecular biology began in 1953 when 'Watson and Francis H. C. Crick proposed that James D. DNA has a double-helical structure. Their proposal, based on analysis of x-ray diffraction patterns generated by Rosalind Franklin and Maurice Wilkins, and coupled with careful model building, proved correct and paved the way for our modern understandingof how DNA functions as the geneticmaterial. 3'end DNA consists of two associatedpolynucleotide strands OH H that wind together to form a double helix. The two sugarA FIGURE 4-2 Chemicaldirectionalityof a nucleicacidstrand. phosphate backbonesare on the outside of the double helix, Shownherearealternative representations of a singlestrandof DNA and the bases project into the interior. The adjoining basesin containing onlythreebases: (C), (A), cytosine adenine andguanine (G) (a)Thechemical each strand stack on top of one another in parallel planes structure showsa hydroxyl groupat the 3, endand (Figure 4-3a). The orientation of the two strands is antipara phosphate groupat the 5, end Notealsothattwo phosphoester bondslinkadjacent nucleotides; allel; that is, their 5'-+3' directions are opposite.The strands thistwo-bondlinkage commonly rs referredto asa phosphodiester bond.(b)In the ,,stick,'diagram are held in precise register by formation of base pairs be(top),thesugars areindicated asvertical linesandthe phosphodiester tween the two strands: A is paired with T through two hybondsasslanting lines;the bases aredenotedbytheirsingle_letter drogen bonds; G is paired with C through three hydrogen abbreviations Inthe simplest (bottom), representation onlythe bases bonds (Figure 4-3b). This base-paircomplementarityis a areindicatedByconvention, a polynucleotide sequence tsatways consequenceof the size,shape,and chemicalcomposition of writtenin the 5'-+3'direction (leftto righ0unless otherwise indicated the bases.The presenceof thousands of such hydrogen bonds in a DNA molecule contributes greaiy to the stability of the double helix. Hydrophobic and van der N7aalsinterpyrimidines, which contain a single ring (see Figue 2-17). actions between the stacked adjacent basepairs further staThree of thesebases-A, G, and C-are found in both DNA bilize the double-helicalstrucure. and RNA; however, T is found only in DNA, and U only in In natural DNA, A always hydrogen bonds with T and RNA. (Note that the single-letterabbreviationsfor these G with C, forming A.T and G.C basepairs, as shown in Figbasesare also commonly usedto denotethe entire nucleotides ure 4-3b. These associations,always between a larger in nucleic acid polymers.) purine and smaller pyrimidine are often called '\X/atsonA single nucleic acid strand has a backboae composedof Crick base pairs. Two polynucleotide strands, or regions repeating pentose-phosphateunits from which the purine thereof, in which all the nucleotides form such base pairs and pyrimidine basesextend as side groups. Like a poiypepare said to be complementary. However, in theory and in tide, a nucleic acid strand has an end-to-endchemical oiiensynthetic DNAs, other base pairs can form. For example, guanine(a purine) could theoreticallyform hydrogenbonds with thymine (a pyrimidine), causing only a minor distortion in the helix. The spaceavailable in the helix also would allow pairing between the two pyrimidines cytosine and thymine. Although the nonstandard G.T and C.T basepairs polynucleotide sequencesare written and read in the 5'--+3, are normally not found in DNA, G.U basepairs are quite direction (from left to right); for example,the sequenceAUG common in double-helical regions that form within otheris assumedto be (5')AUG(3,). As we will see, the 5,_+3, wise single-strandedRNA. Nonstandard base pairs do not directionality of a nucleic acid strand is an important propoccur naturally in duplex DNA becausethe DNA copying 114

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4-3 The DNAdoublehelix.(a)Space< FIGURE fillingmodelof B DNA,the mostcommonformof (lightshades) projectinward DNAin cells.Thebases (darkredand backbones fromthesugar-phosphate blue)of eachstrand,buttheiredgesareaccessible Arrowsindicate throughmajorandminorgrooves. bonds of eachstrand.Hydrogen the 5'-+3'direction betweenthe basesarein the centerof the structure arelinedby potential Themajorandminorgrooves (highlighted bonddonorsandacceptors hydrogen (b) double of DNA structure yellow). Chemical in showsthe two sugarschematic helixThisextended bondingbetween phosphate andhydrogen backbones (a)from basepairs,A T andG C. lPart theWatson-Crick (b) part R E f rom 287i755: R Wingetal, 1980,Nature i983,5ciAm 249:941 Dickerson,

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enzyme, which is described later in this chapter, does not permit them. Most DNA in cells is a right-handed helix. The x-ray diffraction pattern of DNA indicatesthat the stacked bases are regularly spaced0.34 nm apart along the helix axis. The helix makes a complete turn every 3.4 nm; thus there are about 10.1 pairs per turn. This is referredto as the B form of DNA, the normal form present in most DNA stretchesin cells. On the outside of B form DNA, the spacesbetweenthe intertwined strands form two helical grooves of different widths describedas the maior groove and the minor groove (seeFigure 4-3a). As a consequence,the atoms on the edges of each basewithin thesegroovesare accessiblefrom outside the helix, forming two types of binding surfaces.DNA-binding proteins can read the sequenceof basesin duplex DNA by contacting atoms in either the major or the minor grooves. Other structuresof DNA have beendescribedin addition to the major B form. Two of these are compared with B DNA in Figure 4-4. Under laboratory conditions in which most of the water is removed from DNA, the crystallographic structure of B DNA changesto the A form, which is wider and shorter than B DNA, with basepairs tilted rather than perpendicular to the helix axis. RNA-DNA and RNARNA helices exist in this form in cells and in vitro. Short DNA moleculescomposed of alternating purine-pyrimidine nucleotides(especiallyG's and C's) adopt an alternative lefthanded helix configuration instead of the normal righthanded helix. This structure is called Z DNA becausethe basesseemto zigzag when viewed from the side. Some evidence suggeststhat Z DNA may occur in cells, although its function is unknown.

By far, the most important modifications in the structure of standard B form DNA come about as a result of protein binding to specificDNA sequences.Although the multitude of hydrogen and hydrophobic bonds between the basesprovide stability to DNA, the double helix is flexible about its long axis. Unlike the ct helix in proteins (seeFigure 3-4)'

( a )B D N A

( b ) AD N A

( c ) ZD N A

4-4 Modelsof variousknown DNA structures.The A FIGURE whichareon the of thetwo strands, backbones sugar-phosphate (lighter areshownin redandblue;the bases in allstructures, outsrde :10.1 base inward(a)TheB formof DNAhas areoriented shades) apart' pairs nm are 0.34 base stacked turn.Adjacent pairsperhelical A formof DNAhas11 basepairsperturnand (b)Themorecompact to the helixaxis a largetilt of the basepairswith respect exhibits (c)Z DNAisa left-handed doublehelix S T R U C T U RO E F N U C L E I CA C I D S

115

\fhy did DNA evolve to be the carrier of genetic information in cellsas opposedto RNA? The hydrogen at the 2' position in the deoxyriboseof DNA makes it a far more stable molecule than RNA, which instead has a hydroxyl group at the 2' position of ribose (SeeFigure2-16).The 2'-hydroxyl groups in RNA participatein the sloq OH--catalyzedhydrolysis of phosphodiesterbonds at neutral pH (Figure4-6).The absenceof 2'-hydroxyl groups in DNA preventsthis process. Therefore, the presenceof deoxyribosein DNA makes it a more stablemolecule-a characteristiccritical to its function in the long-term storageof geneticinformation.

D N A C a nU n d e r g oR e v e r s i b l e S t r a n dS e p a r a t i o n During replication and transcription of DNA, the strands of the double helix must separateto allow the internal FIGURE 4-5 Proteininteractioncan bend DNA.Theconserved edgesof the basesto pair with the basesof the nucleotides C-terminal domainof theTATAbox-binding protein(TBp) bindsto being polymerizedinto new polynucleotidechains.In later theminorgrooveof specific DNAsequences richin A andl, untwisting sections,we describethe cellular mechanismsthat separate andsharply bending thedoublehelixTranscription of mosteukaryotic and subsequentlyreassociateDNA strandsduring replicagenesrequires participation of TBPlndapted fromD B Nikolov andS K tion and transcription. Here we discuss fundamental Burley,1997, ProcNat'lAcad SciU5A94:151 factors influencing the separation and reassociation of DNA strands.Thesepropertiesof DNA were elucidatedby in vitro expenments. thereare no hydrogenbondsparallelto the axis of the DNA The unwinding and separation of DNA strands,referred helix. This properry allows DNA to bend when complexed to as denaturation, or "melting," can be induced experiwith a DNA-binding protein (Figure4-5). Bendingof DNA mentally by increasing the temperature of a solution of is critical to the dense packing of DNA in chromatin, the DNA. As the thermal energy increases, the resulting protein-DNA complex in which nuclearDNA occursrn euincreasein molecular motion eventually breaks the hydrokaryotic cells(Chapter6). gen bonds and other forces that stabilizethe double helix;

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CHAPTER 4

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B A S I CM O L E C U L A G R E N E T TM CE C H A N T S M S

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FIGURE 4-7 G.Ccontentof DNAaffects EXPERIMENTAL at whichDNAdenatures meltingtemperature.Thetemperature with the proportion of G.Cpairs(a)Meltingof doubledincreases of ultraviolet by theabsorption stranded DNAcanbe monitored DNAunpair, the lightat 260 nm.As regions of double-stranded increases almosttwofoldThe absorption of lightby thoseregions

DNA in a double-stranded at whichhalfthe bases temperature of melting") isdenotedI' (for"temperature havedenatured sample muchlessasthe DNAchanges by single-stranded Lightabsorption (b) of the G'Ccontent function The I. is a isincreased temperature the I. the greater of the DNA;the higherthe G+C percentage,

the strandsthen separate,driven apart by the electrostatic repulsion of the negatively charged deoxyribose-phosphate backbone of each strand. Near the denaturation temperature, a small increasein temperaturecausesa rapid, near simultaneousloss of the multiple weak interactions holding the strands together along the entire length of the DNA molecules.Becausethe stackedbasepairs in duplex DNA absorb lessultraviolet (UV) light than the unstackedbases DNA, this leadsto an abrupt increasein in single-stranded the absorption of UV light, a phenomenon known as hyperchromicity (Figure 4 -7a). The mebing temperature (T-) at which DNA strands will separatedependson severalfactors.Moleculesthat contain a greater proportion of G'C pairs require higher temperatures to denature becausethe three hydrogen bonds in G'C pairs make thesebasepairs more stablethan A'T pairs, which have only two hydrogen bonds. Indeed, the percentage of G'C base pairs in a DNA sample can be estimated from its T- (Figure4-7b\.The ion concentrationalso influences the T- becausethe negatively charged phosphate groups in the two strands are shieldedby positively charged ions. When the ion concentration is low, this shielding is decreased,thus increasingthe repulsive forces between the strandsand reducingthe T-. Agentsthat destabilizehydrogen bonds, such as formamide or urea, also lower the 7-. Finalln extremesof pH denature DNA at low temperature. At low (acid) pH, the basesbecomeprotonated and thus positively charged, repelling one another. At high (alkaline) pH, the baseslose protons and become negatively charged, again repelling one another becauseof the similar charge.In cells,pH and temperatureare,for the most part, maintained. These features of DNA separation are most useful for manipulating DNA in a laboratory setting. The single-strandedDNA molecules that result from denaturation form random coils without an organized

structure. Lowering the temperature, increasing the ion concentration, or neutralizing the pH causes the two complementarystrandsto reassociateinto a perfect double helix. The extent of such renaturation is dependenton time, the DNA concentration, and the ionic concentration. Two DNA strands not related in sequencewill remain as random coils and will not renature; most importantly, they will not inhibit complementary DNA partner strands from finding each other and renaturing. Denaturation and renaturation of DNA are the basis of nucleic acid hybridization, a powerful technique used to study the relatednessof two DNA samplesand to detect and isolate specificDNA moleculesin a mixture containing numerous different DNA sequences ( s e eF i g u r e5 - 1 6 ) .

TorsionalStressin DNA ls Relievedby Enzymes Many prokaryotic genomic DNAs and many viral DNAs are circular molecules. Circular DNA molecules also occur in mitochondria, which are present in almost all eukaryotic cells, and in chloroplasts, which are present in plants and some unicellular eukaryotes' Each of the two strands in a circular DNA molecule forms a closed structure without free ends' Localized unwinding of a circular DNA molecule, which occurs during DNA replication, induces torsional stress into the remaining portion of the molecule becausethe ends of the strands are not free to rotate. As a result, the DNA molecule twists back on itself, like a twisted rubber band, forming supercoils(Figure 4-8a). In other words, when part of the DNA helix is underwound, the remainder of the molecule becomes overwound. Bacterial and eukaryotic cells, however, contain topoisomerase .I, which can relieve any torsional stressthat developsin cellular DNA moleculesduring This enzymebinds to DNA replication or other processes. ACIDS S T R U C T U ROEF N U C L E I C

.

117

(a) Supercoiled

(b) Relaxedcircle

at random sites and breaks a phosphodiesterbond in one strand. Such a one-strand break in DNA is called a nick. The broken end then unwinds around the uncut strand, leadingto loss of supercoils(Figure4-8b). FinallS the same enzyme joins (ligates)the two ends of the broken strand. Another type of enzyme, topoisomerase11,makes breaks in both strands of a double-stranded DNA and rhen religates them. As a result, topoisomerase II can both relieve torsional stressand link together two circular DNA molecules a s i n t h e I i n k so f a c h a i n . Although eukaryotic nuclear DNA is linear,long loops of DNA are fixed in place within chromosomes (Chapter 6). Thus torsional stressand the consequentformation of supercoils also could occur during replication of nuclear DNA. As in bacterial cells, abundant topoisomeraseI in eukaryotic nuclei relieves any torsional stressin nuclear DNA that would develop in the absenceof this enzyme.

DifferentTypesof RNA Exhibit Various ConformationsRelatedto Their Functions The primary structure of RNA is generally similar to that of DNA with two exceptions: the sugar component of RNA, ribose, has a hydroxyl group at the 2, position (seeFigure 2-16b), and thymine in DNA is replaced by uracil in RNA. The presenceof thymine rarher rhan uracil in DNA is important to the long-term stability of DNA becauseof its function in DNA repair (seeSection 4.6). As noted earlier, the hydroxyl group on C2 of ribose makes RNA more chemically labile than DNA. As a result of this labilitS RNA is cleaved into mononucleotides by alkaline solution (see Figure 4-6), whereas DNA is not. The C2 hydroxyl of RNA also provides a chemically reactive group that takes part in 118

.

cHAprER 4

I

< EXPERIMENTAT FIGURE 4-8 Topoisomerase I relievestorsionalstresson DNA.(a)Electron micrograph of SV40viralDNA.Whenthecircular DNAof the SV40virusisisolated andseoarated protein, fromitsassociated the DNAduplexis underwound andassumes thesupercoiled config(b)lf a supercoiled uration. DNAisnicked(i e., onestrandcleaved), the strands canrewind, leading to lossof a supercoil. Topoisomerase I catalyzes thisreaction andalsoreseals the broken endsAll thesupercoils in isolated SV40DNAcan be removed bythe sequential actionof thisenzyme, producing the relaxed-circle conformation For clarity, theshapes of the molecules at the bottom havebeensimplified

RNA-mediated catalysis.Like DNA, RNA is a long polynucleotide that can be double-strandedor single-stranded, linear or circular. It can also participate in a hybrid helix composed of one RNA strand and one DNA strand. As discussedabove, RNA-RNA and RNA-DNA double helices have a compact conformation like the A form of DNA (see Figure4-4b). Unlike DNA, which exists primarily as a very long double helix, most cellular RNAs are single-strandedand exhibit a vaiety of conformations (Figure 4-9). Differences in the sizesand conformations of the various types of RNA permit them to carry out specific functions in a cell. The simplest secondary structures in single-strandedRNAs are formed by pairing of complementary baseswithin a linear sequence."Hairpins" are formed by pairing of baseswithin :5-10 nucleotidesof eachother,and "stem-loops" by pairing of basesthat are separatedby >10 to severalhundred nucleotides.These simple folds can cooperate to form more complicated tertiary structures, one of which is termed a "pseudoknot." As discussedin detail later, IRNA molecules adopt a well-defined three-dimensional architecture in solution that is crucial in protein synthesis.Larger rRNA molecules also have locally well-defined three-dimensional structures, with more flexible links in between.Secondaryand tertiary structures also have been recognized in mRNA, particularly near the ends of molecules. Clearly, then, RNA moleculesare like proteins in that they have structured domains connecred by less structured, flexible stretches. The folded domains of RNA molecules not only are structurally analogous to the a helicesand B strands found in proteins, but in some casesalso have catalytic capacities.

B A s t cM o L E c u L A G R E N E T tMc E c H A N t s M S

In this chapter, we focus on the functions of mRNA, tRNA, and rRNA in gene expression.In later chapters we will encounter other RNAs, often associatedwith proteins' that participate in other cell functions.

(a) Secondarystructure

Structure of NucleicAcids Stem-loop (blTertiarystructure Ca,

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4-9 RNAsecondaryand tertiary structures.(a)StemA FIGURE andothersecondary structures canformby base loops,hairpins, pairingbetweendistantcomplementary of an RNAmolecule. segments loopbetween the base-paired thesingle-stranded In stem-loops, of nucleotrdes or eventhousands helical stemmaybe hundreds in hairpins, theshortturn maycontainasfew asfour long,whereas (b)Pseudoknots, are onetypeof RNAtertiarystructure, nucleotides. loopsthroughbasepairing formedby interaction of secondary shownformsthe core basesThestructure betweencomplementary RNAlefti Secondary-structure domainof the humantelomerase in greenandblueandsinglewith base-paired nucleotides diagram RNA of the telomerase strandedregionsin red.Middle:Sequence diagram to correspond to thesecondary-structure coredomain,colored coredomainstructure at the left.R/ghtiDiagramof the telomerase onlyanda tube bases by 2D-NMR, showingbase-paired determined to the colored to correspond backbone, for the sugar-phosphate (b),middle fromC A Theimer andright,adapted to the left [Part diagrams et al , 2005, Mol Cell 17:671]l

Such catalytic RNAs are called ribozymes. Although ribozymes usually are associatedwith proteins that stabilize the ribozyme structure, it is the RNA that acts as a catalyst. Some ribozymes can catalyzesplicing, a remarkable process in which an internal RNA sequenceis cut and removed, and the two resulting chains then ligated. This process occurs during formation of the majority of functional mRNA moleculesin multicellular eukaryotes,and also occurs in single-celled eukaryotes such as yeast, bacteria, and archaea. Remarkably, some RNAs carry out self-splicing, with the catalytic activity residing in the sequencethat is removed. The mechanismsof splicing and self-splicingare discussedin detail in Chapter 8. As noted later in this chapter, rRNA plays a catalytic role in the formation of peptide bonds during protein synthesis.

r Deoxyribonucleic acid (DNA), the geneticmaterial, carries information to specify the amino acid sequencesof proteins. It is transcribed into severaltypes of ribonucleic acid (RNA), including messengerRNA (mRNA), transfer RNA (IRNA), and ribosomal RNA (rRNA), which function in protein synthesis(seeFigure4-1). r Both DNA and RNA are long' unbranched polymers of nucleotides, which consist of a phosphorylated pentose linked to an organic base,either a purine or pyrimidine' The purines adenine (A) and guanine (G) and the pyrimine cytosine (C) are presentin both DNA and RNA. The pyrimidine thymine (T) present in DNA is replaced by the pyrimidine uracil (U) in RNA. r Adiacent nucleotides in a polynucleotide are linked by phosphodiester bonds. The entire strand has a chemical directionality with 5' and 3' ends (seeFigwe 4-2). r Natural DNA (B DNA) contains two complementary antiparallel polynucleotide strands wound together into a regular right-handed double helix with the baseson the inside and the two sugar-phosphatebackbones on the outside (seeFigure 4-3). Basepairing betweenthe strands and hydrophobic interactions between adjacent base pairs stacked perpendicular to the helix axis stabilize this natlve structure. r The bases in nucleic acids can interact via hydrogen bonds. The standard Watson-Crick base pairs are G'C, A.T (in DNA), and G'C, A'U (in RNA). Basepairing stabilizes the native three-dimensionalstructures of DNA and RNA. r Binding of protein to DNA can deform its helical structure, causing local bending or unwinding of the DNA molecule. r Heat causesthe DNA strandsto separate(denature).The melting temperature7- of DNA increaseswith the percentage of G'C basepairs. Under suitableconditions, separated complementarynucleic acid strandswill renature. r Circular DNA moleculescan be twisted on themselves' forming supercoils (see Figure 4-8). Enzymes called topoisomerasescan relieve torsional stressand remove supercoils from circular DNA molecules' Long linear DNA ian also experiencetorsional stressbecauselong loops are fixed in place within chromosomes' r Cellular RNAs are single-strandedpolynucleotides, some of which form well-defined secondary and tertiary structures (seeFigure 4-9). Some RNAs, called ribozymes' have catalytic activitY.

S T R U C T U RO E F N U C L E I CA C I D S

119

Transcription of Protein-Coding

@

Genesand Formationof Functional mRNA The simplest definition of a gene is a "unir of DNA that contains the information to specify synthesisof a single polypeptide chain or functional RNA (such as a IRNA)." The DNA moleculesof small viruses contain only a few genes,whereas the single DNA molecule in each of the chromosomesof higher animals and plants may contain severalthousandgenes.The vast maiority of genescarry rnformation to build protein molecules,and it is the RNA copies of such protein-coding genes that constitute the mRNA moleculesof cells. During synthesisof RNA, the four-base language of DNA containing A, G, C, and T is simply copied, or transcribed,into the four-baselanguageof RNA, which is identical except that U replacesT. In contrast, during protein synthesis, the four-base language of DNA and RNA is translatedinto the 20-amino acid languageof proteins. In this section,we focus on formation of functional mRNAs from protein-coding genes(seeFigure 4-1, A). A similar process yields the precursors of rRNAs and tRNAs encoded by rRNA and IRNA genes;these precursorsare then further modified to yield functional rRNAs and tRNAs. In addition, thousands of recently discovered micro RNAs (miRNAs) that function ro resulare translat i o n o f s p e c i f i ct a r g e tm R N A s a n d r r a n s c r i p t - i oonf s p e c i f i c target genesare transcribedinto precursorsby RNA polymerasesand processedinto functional miRNAs. Transcription and processingof these other types of RNA is discussed in Chapter 8. Regulation of transcription allows distinct setsof genesto be expressedin the many different types of cellsthat make up a multicellular organism.It also allows different amounrs of mRNA to be transcribedfrom different genes, resulting in differences in the amounts of the encodedproteins in a cell. Regulation of transcription is addressedin Chapter 7.

A TemplateDNA Strand ls Transcribedinto a C o m p l e m e n t a rR y N AC h a i nb y R N Ap o l y m e r a s e During transcription of DNA, one DNA strand acs as a template, determining the order in which ribonucleoside triphosphate (rNTP) monomers are polymerized to form a complementary RNA chain. Basesin the template DNA strand base-pair with complementary incoming rNTps, which then are joined in a polymerization reaction catalyzed by RNA polymerase.Polymerization involves a nucleophilic attack by the 3' oxygen in the growing RNA chain on the ct phosphate of the next nucleotide precursor to be added, resulting in formation of a phosphodiesterbond and release of pyrophosphate(PP;).As a consequenceof this mechanism, RNA moleculesare always synthesizedin the 5,-+3, direction (Figure 4-10a). The energeticsof the polymerization reaction strongly favor addition of ribonucleotides to the growing RNA 120

.

c H A p r E4R |

chain becausethe high-energybond betweenthe cr and B phosphate of rNTP monomers is replaced by the lower-energy phosphodiesterbond betweennucleotides.The equilibrium for the reaction is driven farther toward chain elongation by pyrophosphatase,an enzyme that catalyzescleavageof the releasedPP; into two molecules of inorganic phosphate. Like the two strands in DNA, the template DNA strand and the growing RNA strand that is base-pairedto it have opposite5'-+3' directionality. By convention, the site on the DNA at which RNA polymerasebegins transcription is numbered *1. Downstream denotes the direction in which a template DNA strand is transcribed;upstreamdenotesthe opposite direction. Nucleotide positions in the DNA sequencedownstream from a start site are indicated by a positive (+) sign; those upstream,by a negative(- ) sign. BecauseRNA is synthesized5'-+3', RNA polymerase moves down the template DNA strand in a 3'-+5' direction. The newly synthesizedRNA is complementary to the template DNA strand; therefore, it is identical with the nontemplate DNA strand, except with uracil in place of thymine (see F i g u r e4 - 1 0 b ) . Stages in Transcription To carry out transcription, RNA polymerase performs several distinct functions, as depicted in Figure 4-11. During transcription initiation, RNA polymeraserecognizesand binds to a specific site, called a promoter, in double-strandedDNA (step [). RNA polymerases require various protein factors, called general transcription factors, to help them locate promoters and initiate transcription. After binding to a promoter, RNA polymerase separates the DNA strandsin order to make the basesin the template strand available for basepairing with the basesof the ribonucleosidetriphosphatesthat it will polymerize rogether. RNA polymerasesmelt 12-t4base pairs of DNA around the transcription start site, which is located on the template strand within the promoter region (step [). This allows the template strand to enter the active site of the enzyme that catalyzesphosphodiester bond formation between ribonucleoside triphosphates that are complementary to the promoter template strand at the start site of transcription. The 12-14-base-pairregion of melted DNA in the polymeraseis known as the "transcription bubble." Transcription initiation is considered complete when the first two ribonucleotides of an RNA chain are linked by a phosphodiester bond (stepS). After several ribonucleotides have been polymerized, RNA polymerase dissociatesfrom the promoter DNA and generaltranscription factors.During the stageof strand elongation, RNA polymerasemoves along the template DNA one baseat a time, opening the double-strandedDNA in front of its direction of movement and guiding the strandstogetherso that they hybridize at the upstream end of the transcriprion bubble (Figure4-11, step@). One ribonucleotideat a time is added to the 3' end of the growing (nascent)RNA chain during strand elongation by the polymerase. The enzyme maintains a melted region of approximately 1.4 base pairs, the transcription bubble. Approximately eight nucleotidesat

B A s t cM o L E c u L AGRE N E TM t cE c H A N t s M s

the 3' end of the growing RNA strand remain base-pairedto the template DNA strand in the transcription bubble. The elongation complex, comprising RNA polymerase,template DNA, and the growing (nascent)RNA strand, is extraordinarily stable. For example, RNA polymerasetranscribesthe longestknown mammalian gene,containing about 2 million basepairs, without dissociatingfrom the DNA template or releasingthe nascentRNA. SinceRNA synthesisoccurs at a rate of about 1000 nucleotidesper minute at 37 "C, the elongation complex must remain intact for more than 24 hours to assurecontinuous RNA synthesisof the pre-mRNA from this very long gene. During transcription termination, the final stagein RNA synthesis,the completed RNA molecule, or primary transcript, is releasedfrom the RNA polymerase, and the polymerase dissociatesfrom the template DNA (Figure4-11, step El). Specific sequencesin the template DNA signal the bound RNA polymeraseto terminate transcription. Once released,

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121

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Polymeraseadvances 3'+ 5'down template strand,meltingduplex DNA and addingrNTps to growingRNA. TERMINATION

!

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an RNA polymeraseis free to transcribethe samegeneagain or another gene.

Organizationof GenesDiffers in Prokaryotic a n d E u k a r y o t i cD N A

Structure of RNA Polymerases The RNA polymerases of bacteria, archaea,and eukaryotic cells are fundamentally similar in structure and function. Bacterial RNA polymerasesare composedof two relatedlarge subunits(B, and B), two copiesof a smallersubunit (cr),and one copy of a fifth subunit (o) that is not essentialfor transcription or cell viability but stabilizesrhe enzyme and assisrsin the assembly of its subunits. Archaeal and eukaryotic RNA polymerases have several additional small subunits associated with this core complex, which we describe in Chapter 7. Schematic diagrams of the transcription process generally show RNA polymerasebound to an unbent DNA molecule, as in Figure 4-11. However, X-ray crystallography and other studies of an elongating bacterial RNA poiymerase indicate that the DNA bends at rhe transcription bubble (Figwe 4-12).

Having outlined the process of transcription, we now briefly consider the large-scalearrangement of information in DNA and how this arrangement dictates the requirementsfor RNA synthesisso that information transfer goes smoothly. In recent years, sequencingof the entire genomes from several organisms has revealed not only large variations in the number of protein-codinggenesbut also differencesin their organization in prokaryotes and eukaryotes. The most common arrangementof protein-coding genes in all prokaryotes has a powerful and appealing logic: genes encoding proteins that function together, for example, the enzymesrequired to synthesizethe amino acid tryptophan, are most often found in a contiguous array in the DNA. Such an arrangement of genesin a functional group is called an operon becauseit operatesas a unit from a singlepromoter. Transcription of an operon produces a continuous strand of

122

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BASTC MOLECULAG R E N E T TM CE C H A N T S M S

mRNA that carriesthe messagefor a relatedseriesof proteins (Figure 4-13a). Each section of the mRNA representsthe unit (or gene)that encodesone of the proteins in the series. This arrangementresults in the coordinate expressionof aIl the genes in the operon. Every time an RNA polymerase molecule initiates transcription at the promoter of the operon, all the genesof the operon are transcribedand translated. In prokaryotic DNA the genesare closelypacked with very few noncoding gaps, and the DNA is transcribed directly into mRNA. BecauseDNA is not sequesteredin a nucleus in prokaryotes, ribosomes have immediate accessto the translation start sitesin the mRNA as they emergefrom the surface of the RNA polymerase. Consequently,translat i o n o f t h e m R N A b e g i n se v e n w h i l e t h e 3 ' e n d o f t h e mRNA is still being synthesizedat the active site of the

(a)

(b)

B subunit..r

-d ':' B'subunit/

r-1

IRNAZ n subunit

\o

subunit

Thisstructure corre4-12 BacterialRNApolymerase. A FIGURE phase(step4) molecule in theelongation to the polymerase sponds in the is proceeding transcriptton of Figure4-11 ln thesediagrams, DNAenters wheredownstream leftwarddirectionArrowsindicate andupstream DNAexitsat an anglefromthe downthe polymerase strandblue, streamDNA;thecodingstrandisred,the noncoding gold,B islight is subunit RNAgreenTheRNApolymerase nascent B' gray,andthe o subunitvisible fromthisangleisbrown In (a)a froman isvrewed modelof theelongation complex space-filling throughthe the bendin the DNAasit passes anglethatemphasizes polymerase is rotatedin (b)asshown,and Theelongation complex of the the structure proteins transparent to reveal aremadelargely in the that isnotvisible the polymerase bubbleinside transcription DNA to thetemplate modelNucleotides complementary space-filling RNAstrand(atthe left) The areaddedto the 3'-endof the nascent at the bottom nascent RNAexitsthe polymerase newlysynthesized a c h a n n ef ol r m e db e t w e etnh eB a n dB ' s u b u n i tTs h et r through fromthisangle[Courtesy subunitandtheothercrsubunitarevisible o f S e t hD a r s t ;s e eN K o r z h e v ae t a l , 2 0 0 0 , S c i e n c e 2 8 9 , 6 1 9 - 6 2 5a,n d N Opalkaet al , 2003, Cell'114:335-345I

RNA polymerase. This economic clustering of genes devoted to a single metabolic function does not occur in eukaryotes, even simple ones like yeasts,which can be metabolically similar to bacteria. Rather, eukaryotic genes encoding proteins that function together are most often physically separatedin the DNA; indeed such genes usually are located on different chromosomes.Each gene is transcribed from its own promoter, producing one mRNA, which generally is translated to yield a singlepolypeptide (Figure 4-13b). When researchersfirst compared the nucleotide sequences of eukaryotic mRNAs from multicellular organisms with the DNA sequencesencoding them, they were surprised to find that the uninterrupted protein-coding sequenceof a given mRNA was discontinuous in its corresponding section of DNA. They concluded that the eukaryotic gene existed in pieces of coding sequence,the exons, separated by non-protein-coding segments' the introns. This astonishingfinding implied that the long initial primary transcript-the RNA copy of the entire transcribed DNA sequence-had to be clipped apatt to remove the introns and then carefully stitched back together to produce eukaryotic mRNAs. Although introns are common in multicellular eukaryotes, they are extremely rare in bacteria and archaeaand uncommon ln many unicellular eukaryotes such as baker's yeast. However, introns are present in the DNA of viruses that infect eukaryotic cells. Indeed, the presenceof introns was first discovered in such viruses, whose DNA is transcribedby host-cellenzymes.

EukaryoticPrecursormRNAsAre Processed l RNAs t o F o r mF u n c t i o n am In prokaryotic cells, which have no nuclei, translation of an mRNA into protein can begin from the 5' end of the mRNA evenwhile the 3' end is still being synthesizedby RNA polymerase,In other words, transcription and translation occur concurrently in prokaryotes. In eukaryotic cells, however, not only is the site of RNA synthesis-the nucleusseparatedfrom the site of translation-the cytoplasm-but also the primary transcripts of protein-coding genes are precursor mRNAs (pre-mRNAs) that must undergo several

LRNA O F F U N C T I O N AM GG E N E SA N D F O R M A T I O N ON F PROTEIN.CODIN TRANSCRIPTIO

123

(a)Prokaryotes

(b) Eukaryotes Yeastchromosomes Kb

TRP|

TRP4

E. coli genome 580

V

trp operon

-

:

I

E I p I c I B l A -8kb

910 Vll

Staft site for trp mRNA synthesis

680

Xl

rranscrintion f frp mRNA

5'

fff++

trp mRNAs

Start sitesfor protein synthesis I Translation

:Proteins

v -j-

I

-a B

Proteins

1 r

Translation

2345 IIII

FIGURE 4-13 Geneorganizationin prokaryotesand eukaryotes.(a)Thetryptophan(trp)operonisa continuous segment of the E.colichromosome, containing fivegenes(blue) that encodethe enzymes necessary for the stepwise synthesis of tryptophanTheentireoperonistranscribed fromonepromoter into onelongcontinuous frp mRNA(red)Translation of thismRNAbegins at fivedifferent startsites,yielding (green)Theorderof frveproteins the genesin the bacterial genomeparallels the sequential functionof

proteins theencoded (b)Thefivegenes in thetryptophan pathway. encoding theenzymes required for tryptophan synthesis in yeast (Saccharomyces cerevisiae) arecarriedon four differentchromosomes Eachgeneistranscribed fromitsown promoter to yielda primary transcript thatisprocessed intoa functional mRNAencoding a singleprotein. Thelengths of thevarious chromosomes areqivenin (103bases) kilobases

modifications, collectively termed RNA processing,to yield a functional mRNA (seeFigure 4-I,2). This mRNA then must be exported to the cytoplasm before it can be translated into protein. Thus transcription and translation cannot occur concurrently in eukaryotic cells. All eukaryotic pre-mRNAs initially are modified at the two ends, and these modifications are retained in mRNAs. As the 5' end of a nascent RNA chain emergesfrom the surface of RNA polymerase, it is immediately acted on by several enzymes that together synthesize the 5, cap, a 7-methylguanylate that is connected to the terminal nu-

yeastsand invertebratesthan in vertebrates.Poly(A) polymeraseis part of a complex of proteins that can locate and cleavea transcript at a specific site and then add the correct number of A residues,in a processthat does not require a template. The final step in the processing of many different eukaryotic mRNA molecules is RNA splicing: the internal cleavageof a transcripr to excisethe introns, followed by ligation of the coding exons. Figure 4-15 summarizesthe basic steps in eukaryotic mRNA processing, using the B-globin gene as an example. rJTeexamine the cellular machinery for carrying out processing of mRNA, as well as IRNA and rRNA, in Chapter 8. The functional eukaryotic mRNAs produced by RNA processingretain noncoding regions,referred to as 5' and 3, wntranslated regions (UTRs), at each end. In mammalian mRNAs, the 5' UTR may be a hundred or more nucleotides long, and the 3'UTR may be severalkilobasesin length. Prokaryotic mRNAs also usually have 5' and 3' UTRs, but these are much shorter than those in eukaryotic mRNAs. generally containing fewer than 10 nucleotides.

Processingat the 3' end of a pre-mRNA involvescleavage by an endonuclease to yield a free 3,-hydroxyl group to which a string of adenylic acid residuesis added orr. ut " time by an enzyme called poly(A) polymerase. The resulting poly(A) tail contains 100-250 bases,being shorter in 124

.

c H A p r E R4

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B A s t cM o l E c u L A RG E N E I c M E C H A N t s M s

7-Methylguanylate

4-14 Structureof the 5' methylatedcap' Thedistin< FIGURE capon eukaryotic guishing features of the 5' methylated chemical to the initial of 7-methylguanylate mRNAare(1)the 5'-+5' linkage and(2)the methylgroupon the 2' of the mRNAmolecule nucleotide (base1) Boththesefeahydroxyl of the riboseof thefirstnucleotide yeasts plants; lack of higher in cells cells and all animal in turesoccur nucleotide of thesecond 1 Theribose themethylgroupon nucleotide 1976,Cell (base2) alsoismethylated in vertebrates [SeeAJ Shatkin, 9:645I

HH I

o-P:o I o - o - PI: o

l2'

A l t e r n a t i v eR N AS p l i c i n gI n c r e a s e s the Numberof ProteinsExpressed f r o m a S i n g l eE u k a r y o t i cG e n e

OH OH 5' -> 5'linkage

o I o-P:o I o

In contrast to bacterial and archaealgenes,the vast majority of genesin higher, multicellular eukaryotescontain multiple introns. As noted in Chapter 3' many proteins from higher eukaryoteshave a multidomain tertiary structure (seeFigure 3-11). Individual repeatedprotein domains often are encoded by one exon or a small number of exons that code for identical or nearly identical amino acid sequences'Such repeated exons are thought to have evolved by the accidental multiple duplication of a length of DNA lying between two sites in adiacent introns, resulting in insertion of a string of repeated exons) separatedby introns, between the original two introns. The presenceof multiple introns in many eukaryotic genespermits expressionof multiple' related proteins from a single gene by means of alternative splicing. In higher eukaryotes,alternativesplicing is an important mechanism for production of different forms of a protein' called isoforms, by different types of cells'

Base 1 1',

2'

H

o-cH3

-o-P:o

o I

,ro

o-cH3

O-P:O

I

illll}

overview Animation: Life

le of an mRNA

RNAprocessing 4-15 Overviewof RNAprocessing. > FIGURE genecontains produces mRNAin eukaryotes. TheB-globin functional exons(constituting thecodingregion,red)and threeprotein-coding (blue)Theintronsinterrupt the noncoding introns two intervening for aminoacids31 and protein-coding sequence between thecodons genes protein-coding of eukaryotic 32 and105 and106.Transcription thatencodes thefirstaminoacidand startsbeforethesequence encoding thelastaminoacid,resulting beyond thesequence extends (gray) These transcript. in noncoding regions at theendsof theprimary (UTRs) The5' cap regions areretained duringprocessing. untranslated (mtGppp) RNAtranscript, isaddedduringformation of theprimary at the poly(A) whichextendsbeyondthe poly(A)site Aftercleavage removes of multiple A residues to the3' end,splicing siteandaddition referto positions Thesmallnumbers andjoinstheexons. the introns of B-globin. acidsequence in the 147-amino

p-Globin genomrc DNA Start site for

106

147

Poly(A) site

RNA synthesis Primary 5' RNA transcnpt

Aln

p-Globin mRNA

A),

MRNA T R A N S C R I P T I OONF P R O T E I N - C O D I NGGE N E SA N D F O R M A T I O NO F F U N C T I O N A L

125

F i b r o n e c t ig ne n e

Fibroblast f i b r o n e c t i nm R N A Hepatocyte fibronectinmRNA

A FIGURE 4-16 Alternativesplicing.The=75-kbfibronectin gene (fop)contains multiple exons; splicing of frbronectin varies by cell type TheElllBandElllAexons(green) encodebindingdomains for proteins specific on thesurface of fibroblasts. Thefibronectin mRNA Fibronectin, a multidomain protein found in mammals, providesa good exampleof alternativesplicing(Figure4-16). Fibronectin is a long, adhesiveprotein secretedinto the extracellular spacethar can bind other proteins together.\7hat and where it binds dependson which domains are splicedtogether. The fibronectin gene contains numerous exons, grouped into several regions corresponding to specific domains of the protein. Fibroblasts produce fibronectin mRNAs that contain exons EIIIA r.rd EtIIn; these exons encode amino acid sequencesthat bind tightly to proteins in the fibroblast plasma membrane. Consequently,this fibronectin isoform adheresfibroblasrsto the extiacellular matrix. Alternative splicing of the fibronectin primary transcript in hepatocytes,the major type of cell in the liver, yields mRNAs that lack the EIIIA and EIIIB exons. As a result, the fibronectin secretedby hepatocyresinto the blood does not adhere tightly to fibroblasts or most other cell types, allowing it to circulate. During formation of blood clots, however, the fibrin-binding domains of hepatocyte fibronectin binds to fibrin, one of the principal constituentsof clots. The bound fibronectin then interacts with integrins on the membranesof passingplatelets,thereby expanding the clot by addition of platelets. More than 20 different isoforms of fibronectin have been identified, each encoded by a different, alternatively spliced mRNA composed of a unique combination of fibronectin gene exons. Recent sequencingof large numbers of mRNAs isolated from various tissues and comparison of their sequenceswith genomic DNA has revealeJthat nearly 60 percent of all human genesare expressedas alternativelyspliced mRNAs. Clearly, alternative RNA splicing greatly expands the number of proteins encoded by the genomesof higher, multicellular organisms.

Transcriptionof Protein-CodingGenes and Formation of Functional mRNA Transcription of DNA is carried out by RNA polymerase, hich adds one ribonucleotide ar a time to the 3, end of a growing RNA chain (seeFigure4-11). The sequenceof the template DNA strand determines the order in which ribonucleotidesare polymerized to form an RNA chain. 126

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produced in fibroblasts includes the ElllAandElllBexons, whereas theseexonsarespliced out of fibronectin mRNAin hepatocytes. In thisdiagram, introns(blacklines) arenot drawnto scale; mostof themaremuchlongerthananyof theexons. r During transcription initiation, RNA polymerase binds to a specific site in DNA (the promoter), Iocally melts the double-strandedDNA to reveal the unpaired template strand, and polymerizes the first two nucleotides complementary to the template strand. The melted region of t2-14 basepairs is known as the "transcription bubble." r During strand elongation, RNA polymerase moves down the DNA, melting the DNA aheadof the polymerase, so that the template strand can enter the active site of the enzyme,and allowing the complementary DNA strands of the region just transcribed to reanneal behind it. The transcription bubble moves with the polymeraseas the enzyme adds ribonucleotidescomplementaryto the template strand to the 3' end of the growing RNA chain. 'S7hen r RNA polymerasereachesa termination sequence in the DNA, the enzymestops rranscription, leading to releaseof the completed RNA and dissociationof the enzyme from the template DNA. r In prokaryotic DNA, severalprotein-coding genescommonly are clustered into a functional region, an operon, which is transcribed from a single promoter into one mRNA encoding multiple proteins with related functions (seeFigure 4-13a). Translation of a bacterial mRNA can begin before synthesisof the mRNA is complete. r In eukaryotic DNA, each protein-coding gene is transcribedfrom its own promoter.The initial primary transcript very often containsnoncoding regions(introns) interspersed among coding regions (exons). r Eukaryotic primary transcripts must undergo RNA processingto yield functional RNAs. During processing,the ends of nearly all primary transcripts from protein-coding genesare modified by addition of a 5' cap and 3' poly(A) tail. Transcripts from genescontaining introns undergo splicing,the removal of the introns and joining of the exons (seeFigure4-15). r The individual domains of multidomain proteins found in higher eukaryotesare often encodedby individual exons or a small number of exons. Distinct isoforms of such proteins often are expressedin specific cell types as the result of alternative splicing of exons.

BASTC M O L E C U L AG R E N E T TM CE C H A N T S M S

The Decodingof mRNAby tRNAs

!f,

Although DNA storesthe information for protein synthesis and mRNA conveysthe instructions encodedin DNA, most biological activitiesare carried out by proteins.As we saw in Chapter 3, the linear order of amino acidsin eachprotein determinesits three-dimensionalstructure and activity. For this reason,assemblyof amino acids in their correct order, as encoded in DNA, is critical to production of functional proteins and hence the proper functioning of cells and organrsms. Translation is the whole processby which the nucleotide sequenceof an mRNA is used as a template to join the amino acids in a polypeptide chain in the correct order (see Figure 4-1,,g).In eukaryoticcells,protein synthesisoccurs in the cytoplasm, where three types of RNA moleculescome together to perform different but cooperative functions (Figue 4-1.7)z 1,. MessengerRNA (mRNA) carries the geneticinformation transcribed from DNA in a linear form. The mRNA is read in setsof three-nucleotidesequences,called codons, each of which specifiesa particular amino acid. 2. Transfer RNA (IRNA) is the key to decipheringthe codons in mRNA. Each type of amino acid has its own subset of tRNAs, which bind the amino acid and carry it to the growing end of a polypeptide chain when the next codon in

aa7-tRNA7 arflvrng

Growing polypeptide chain

H

Hrru-J-R, H-9

!--o o

?=o

I

o o

tRNA4 leaving

mRNA \____YJ

gYJ

q/J

Codon Codon Codon d0t

ddZ

ElOg

M o v e m e n to f r i b o s o m e

a FIGURE 4-17 The three rolesof RNAin protein synthesis. RNA(mRNA) istranslated intoproteinbythejointaction Messenger of whichiscomposed RNA(IRNA) andthe ribosome, of transfer proteins molecules RNA(rRNA) andtwo majorribosomal numerous (notshown)Notethe basepairingbetween and IRNAanticodons of a peptidebond codonsin the mRNA.Formation complementary aa-tRNA andthe the amino-group N on the incoming between iscatalyzed C on the growingproteinchain(green) carboxy-terminal from aa : aminoacid;R : sidegroup[Adapted by oneof the rRNAs. A J F G r i f f i t h s e t a l , 1 9 9 9 ,M o d e r n G e n e t i cA n a l y s i s , WH F r e e m a na n d C o m p a n yl

the mRNA calls for it. The correct IRNA with its attached amino acid is selectedat each step becauseeach specific IRNA molecule contains a three-nucleotidesequence,an anticodon, that can base-pairwith its complementary codon in the mRNA. 3. Ribosomal RNA (rRNA) associateswith a set of proteins to form ribosomes.Thesecomplex structures,which physically move along an mRNA molecule' catalyzethe assembly of amino acids into polypeptide chains. They also bind tRNAs and various accessoryproteins necessaryfor protein synthesis.Ribosomesare composedof a large and a small subunit, each of which contains its own rRNA molecule or molecules. Thesethree types of RNA participate in the synthesisof proteins in all organisms. Indeed, development of three functionally distinct RNAs was probably the molecular key to the origin of life. In this section,we focus on the decoding of mRNA by IRNA adaptors, and how the structure of each of these RNAs relates to its specific task. How they work together with rRNA, ribosomes, and other protein factors to synthesizeproteins is detailed in the following section.Since translation is essentialfor protein synthesis,the two processes commonly are referred to interchangeably.However, the polypeptide chains resulting from translation undergo posttranslational folding and often other changes(e.g.,chemical modifications, association with other chains) that are required for production of mature' functional proteins (Chapter3).

MessengerRNACarriesInformation from DNA in a Three-LetterGeneticCode As noted above, the genetic code used by cells is a triplet code, with every three-nucleotidesequence'or codon, being "read" from a specifiedstarting point in the mRNA. Of the 54 possiblecodons in the geneticcode' 51 specify individual amino acids and three are stop codons. Table 4-1 shows that most amino acids are encoded by more than one codon. and tryptophan-have a single Only two-methionine codon; at the other extreme, leucine,serine,and arginine are each specifiedby six different codons. The different codons for a given amino acid are said to be synonymous.The code itself is termed degenerate,meaning that a particular amino acid can be specifiedby multiple codons. Synthesisof all polypeptide chains in prokaryotic and eukaryotic cells begins with the amino acid methionine. In bacteria, a specialized form of methionine is used with a formyl group linked to its amino group. In most mRNAs, the start (initiator) codon specifying this amino-terminal methionine is AUG. In a few bacterial mRNAs, GUG is used as the initiator codon, and CUG occasionallyis used as an initiator codon for methionine in eukaryotes. The three codons UAA, UGA, and UAG do not specify amino acids but, rather, constitute stop (termination) codons that mark the carboxyl terminus of polypeptide chains in almost all cells. The sequenceof codons that runs from a specific start T H E D E C O D I N GO F m R N A B Y t R N A s

127

sEcoN0 P0stTt0N

U

E

U

C

A

G

Phe

Ser

Tyr

cys

U

Phe

Ser

Tyr

cyt

C

Leu

Ser

Stop

Stop

A

Leu

Ser

Stop

Ttp

G

Leu

Pro

His

Atg

U

Leu

Pro

His

Atg

C

Leu

Pro

Gln

Arg

Leu (Met)'*

Pro

Gln

Arg

A = G

Ile

Thr

Asn

Ser

U

Ile

Thr

Asn

Ser

C

Ile

Thr

Lys

Atg

A

Met (Start)

Thr

Lys

Arg

G

Val

Ala

Asp

Glv

U

Val

Ala

Asp

Glv

C

Val

Ala

Glu

Glv

A

Val (Met)"

Ala

GIu

Glv

G

z. g I

F a e 4 @ E g

A

G

-E E o q

= o m

*AUG is the most common initiator codon; GUG usually codes for valine and CUG for leucine, but. rarely, these codons can also code for methionine to initiate a protein chain.

codon to a stop codon is called a reading frame. This precise linear array of ribonucleotides in groups of three in mRNA specifiesthe precise linear sequenceof amino acids in a polypeptide chain and also signals where synthesis of the chain starts and stops. Becausethe genetic code is a non-overlapping triplet code without divisions between codons, a particular mRNA theoretically could be translated in three different reading frames. Indeed some mRNAs have been shown to contain overlapping information that can be translatedin different reading frames, yielding different polypeptides (Figure 4-18). The vast majority of mRNAs, however,can be read in only one frame becausestop codonsencountered in the other two possiblereading frames terminatetranslation before a functional protein is produced. Very rarely,

128

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another unusual coding arrangement occurs becauseof frame-shiftins. In this case the protein-synthesizingmachinery may read four nucleotidesas one amino acid and then continue reading triplets, or it may back up one base and read all succeedingtriplets in the new frame until termination of the chain occurs. Only a few dozen such instancesare known. The meaning of each codon is the same in most known organisms-a strong argument that life on earth evolved only once. In fact, the geneticcode shown in Table 4-1 is known as the uniuersal code. However, the geneticcode has beenfound to differ for a few codons in many mitochondria, in ciliated protozoans, and in Acetabwlaria, a single-celled plant. As shown in Table 4-2, most of thesechangesinvolve reading of normal stop codons as amino acids, not an

BASTC M O L E C U L AG R E N E T TM CE C H A N T S M S

(Figure 4-19). The anticodon in the IRNA then base-pairs with a codon in mRNA so that the activatedamino acid can be added to the growing polypeptide chain (seeFigures4-17 Polypeptidel and4-18). Some 30-40 different tRNAs have been identified in Frame2 bacterialcells and as many as 50-100 in animal and plant cells.Thus the number of tRNAs in most cellsis more than 2 Polypeptide the number of amino acids used in protein synthesis(20) and also differs from the number of amino acid codons in 4-18 Multiplereadingframesin an mRNA FIGURE the genetic code (61). Consequently,many amino acids lf translation shownbeginsat two sequence. of the mRNAsequence have more than one IRNA to which they can attach (exupstream startsites(notshown), thentwo overlapping different plaining how there can be more tRNAs than amino acids); framesarepossible In thisexample, thecodonsareshifted reading in addition, many tRNAs can pair with more than one the same onebaseto the rightin the lowerframeAs a result, nucleotide sequence specifies different aminoacidsduringtranslation, codon (explaining how there can be more codons than tRNAs). in two of thethree Althoughregions of sequence thataretranslated The function of IRNA molecules,which are 70-80 nupossible framesarerare,thereareexamples in bothprokaryreading wherethe same cleotideslong, dependson their precisethree-dimensional andespecially in theirviruses, otesandeukaryotes, fromthe same isusedin two alternative mRNAs expressed sequence structures.In solution, all IRNA moleculesfold into a sim, n dt h es e q u e n ci ser e a di n o n er e a d i nfgr a m ei n region o f D N Aa ilar stem-loop arrangement that resemblesa cloverleaf o n em R N Aa n di n a n a l t e r n a t i vr eea d i nfgr a m ei n t h eo t h e rm R N A . when drawn in two dimensions (Figure 4-20a)' The four is wherethe sameshortsequence Thereareevena few instances stems are short double helicesstabilizedby Watson-Crick reading frames readin allthreepossible basepairing; three of the four stemshave loops containing sevenor eight basesat their ends,while the remaining,unloooed stem contains the free 3' and 5' ends of the chain. exchangeof one amino acid for another.Theseexceptionsto The three nucleotidescomposingthe anticodon are located the universalcode probably were later evolutionary developat the center of the middle loop' in an accessibleposition ments; that is, at no single time was the code immutably that facilitates codon-anticodon base pairing. In all tRNAs, fixed, although massivechangeswere not tolerated once a the 3' end of the unlooped amino acrdacceptor stem has the generalcode began to function early in evolution. sequenceCCA, which in most casesis added after synthesis and processingof the IRNA are complete.Severalbasesin The FoldedStructureof tRNA Promoteslts most tRNAs also are modified after transcription, creating D e c o d i n gF u n c t i o n s nonstandard nucleotidessuch as inosine, dihydrouridine, and pseudouridine. As we will see shortly, some of these Translation, or decoding, of the four-nucleotidelanguageof modified basesare known to play an important role in proDNA and mRNA into the 2O-amino acid languageof protein synthesis. Viewed in three dimensions, the folded teins requires tRNAs and enzymes called aminoacyl-tRNA IRNA molecule has an L shape with the anticodon loop synthetases.To participate in protein synthesis,a IRNA moland acceptor stem forming the ends of the two arms (Figecule must become chemically linked to a particular amino ure4-20b\. acid via a high-energy bond, forming an aminoacyl-tRNA F r a m e1

C()DON

Uf'IIVERSAL C0DE

C(]DTUNUSUAL

OCCURRENCE

UGA

Stop

Trp

My cop lasma, Spir op lasma, mitochondria of many species

CUG

Leu

Thr

Mitochondria in yeasts

UAA, UAG

Stop

Gln

A cetab ul ar ia, Tetr ah y m ena, Paramecium, etc.

UGA

Stop

cys

Euplotes

"Found in nuclear genesof the listed organisms and in mitochondrial genesas indicated. souRCE:S. Osawa et al., 1.992,Microbiol. Reu. 56:229.

OF mRNABY tRNAs THEDECODING

129

A m i n o a c i d( P h e )

High-energy ester bond

HO

til

H 2 N- C - C

-OH

r'',li,,

d

z

E Linkaqe of P;t" 1i 131114Ptre

ATP AAA AminoacyltRNA synthetase tRNA specificfor specificfor Phe Phe (tRNAPhe)

AMP + PPt AAA Aminoacyl-tRNA

FIGURE 4-19 Decodingnucleicacidsequenceinto amino acidsequence. Theprocess for translating nucleic acidsequences in mRNAintoaminoacidsequences in proteins involves two steps. Step[: An aminoacyl-tRNA synthetase f irstcouples a specific amino acid,viaa high-energy esterbond(yellow), to eitherthe 2, or 3,

AAA mRNA

hydroxyl of theterminal adenosine in the corresponding IRNA. StepE: A three-base sequence in theIRNA(theanticodon) then base-pairs with a codonin the mRNAspecifying theattached amino acid.lf an erroroccurs in eitherstep,thewrongaminoacidmaybe incorporated intoa polypeptide chainPhe: phenylalanine

NonstandardBasePairingOften Occurs BetweenCodonsand Anticodons

@ = dihYdrouridine = inosine e = ribothymidine e

If perfect Watson-Crick base pairing were demanded between codons and anticodons,cellswould have to contain at least 61 different types of tRNAs, one for each codon that specifiesan amino acid. As noted above,however,many cells contain fewer than 61 tRNAs. The explanation for the smaller number lies in the capability of a single IRNA anticodon to recognizemore than one, but not necessarilyeverg codon corresponding to a given amino acid. This broader recognition can occur becauseof nonstandard pairing between bases in the so-called uobble position: that is, the third (3') base in an mRNA codon and the corresponding first (5') basein its IRNA anticodon. The first and second basesof a codon almost always form standard Watson-Crickbasepairs with the third and secondbases,respectively,of the correspondinganticodon, but four nonstandardinteractionscan occur betweenbases in the wobble position. Particularly important is the G.U base pair, which structurally fits almost as well as the standard G.C pair. Thus, a given anticodon in IRNA with G in the first (wobble) position can base-pairwith the two

= pseudouridine @ m = methylgroup

T{/CG loop

Net result: Phe is selected by its codon

P h e - t R N A P h eb i n d s to the UUU codon

Acceptor stem

< FIGURE 4-20 Structureof tRNAs.(a)Althoughtheexact nucleotide sequence varies amongtRNAs, theyallfold intofour base-paired stemsandthreeloopsTheCCAsequence at the 3, end alsoisfoundin alltRNAsAttachment of an aminoacidto the 3, A yieldsan amrnoacyl-tRNA. Someof theA, C, G, andU residues are post-transcriptionally modified (seekey).Dihydrouridine in mosttRNAs (D)is nearlyalwayspresent in the D loop;likewise, (T) ribothymidine (V) arealmostalwayspresent andpseudouridlne in theTTITCG loop. Yeast alanine IRNA,represented here,alsocontains othermodified basesThetripletat thetip of theanticodon loopbase-pairs with the corresponding codonin mRNA(b)Three-dimensional modelof the generalized backbone of alltRNAsNotethe L shapeof the molecule. [Part(a) see R W Hollyet al , 1965, Science147i1462;part (b) from J G Arnez and D Moras, 1997, TrendsBiochem Sci 22:211 I

130

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B A S | CM O L E C U L A G R E N E T TM CE C H A N T S M S

tRNA

lf these basesare in first, or wobble, positionof a nticodon

321 12 5 ' m R N A3 '

5' mRNA 12 321

t h e nt h e t R N A m a y recognizecodons in m R N A h a v i n gt h e s e b a s e si n t h i r d p o s i t i o n

nized by the sameIRNA with the anticodon 3'-GAI-S'; the inosine in the wobble position forms nonstandard base pairs with the third basein the four codons. In the caseof in. UUR codon, a nonstandard G'U pair also forms between position 3 of the anticodon and position 1 of the codon.

Amino Acids BecomeActivatedWhen CovalentlyLinkedto tRNAs Recognition of the codon or codons specifying a given amino acid by a particular IRNA is actually the secondstep in decoding the genetic message.The first step' attachment

lf these basesare in t h i r d ,o r w o b b l e ,p o s i t i o n of codon of an mRNA then the codon may be recognizedby a tRNA havingthese basesin first position of anticodon

the 3' terminus of IRNA moleculesby an AlP-requiring reaction. In this reaction, the amino acid is linked to the tRNA by a high-energybond and is thus said to be actiuated. The energy of this bond subsequentlydrives formation of the peptide bonds linking adjacent amino acids in a growing c poiypeptide chain. The equilibrium of the aminoacylation 3', i.u.tiotr is driven further toward activation of the amino tRNA acid by hydrolysis of the high-energyphosphoanhydride 4-21 Nonstandard basepairingat the wobble A FIGURE bond in the releasedpyrophosphate (seeFigute 4-19). of an mRNA position.Thebasein thethird(orwobble)position Aminoacyl-IRNA synthetasesrecognize their cognate basepairwith the basein thefirst codonoftenformsa nonstandard tRNAs by interacting primarily with the anticodon loop (orwobble)position of a tRNAanticodonWobblepairingallowsa and acceptor stem, although interactions with other reit morethanone mRNAcodon(top);conversely, IRNAto recognize gions of a IRNA also contribute to recognition in some by morethanonekindof IRNA allowsa codonto be recognized cases.Also, specific bases in incorrect tRNAs that are (bottom), eachIRNAwill bearthesameaminoacid Note although structurally similar to a cognate IRNA will inhibit chargcan"read"(become in thewobbleposition thata IRNAwith l(inosine) ing of the incorrect IRNA. Thus, recognition of the correct pairedwith)threedifferent codons, anda IRNAwith G or U in the tRNA dependson both positive interactions and the abcanreadtwo codonsAlthoughA istheoretically wobbleposition senceof negative interactions. Still, becausesome amino possible it isalmostnever of the anticodon, in thewobbleposition acids are so similar structurally, aminoacyl-tRNA synfoundin nature thetasessometimesmake mistakes. These are corrected' however, by the enzymesthemselves,which have a proofreading activity that checks the fit in their amino correspondingcodonsthat have either pyrimidine (C or U) acid-binding pocket. If the wrong amino acid becomesatin the third position (Figure4-21).For example,the phenytached to a tRNA, the bound synthetase catalyzesremoval lalanine codons UUU and UUC (5'-+3') are both recogof the amino acid from the IRNA' This crucial function nized by the IRNA that has GAA (5'-+3') as the anticodon. : helps guarantee that a IRNA delivers the correct amino any base; In fact, any two codons of the type NNPyr (N : acid t" the protein-synthesizingmachinery. The overall pyrimidine) encode a single amino acid and are Pyr error rate for translation in E' coli is very loq approxidecoded by a single tRNA with G in the first (wobble) mately 1 per 50,000 codons, evidenceof both the fidelity position of the anticodon. of IRNA iecognition and the importance of proofreading Although adenine rarely is found in the anticodon by aminoacyl-IRNA synthetases. wobble position, many tRNAs in plants and animals conthis tain inosine (I), a deaminatedproduct of adenine, at position. Inosine can form nonstandardbasepairs with A, C, and U. A IRNA with inosine in the wobble position thus can recognizethe correspondingmRNA codons with The Decoding of mRNA bY tRNAs A , C , o r U i n t h e t h i r d ( w o b b l e )p o s i t i o n ( s e eF i g u r e4 - 2 1 ) ' r Genetic information is transcribed from DNA into mRNA For this reason,inosine-containingtRNAs are heavily emin the form of an overlapping,degeneratetriplet code' ployed in translation of the synonymouscodons that specify r Each amino acid is encoded by one or more threea single amino acid. For example, four of the six codons nucleotide sequences(codons) in mRNA. Each codon for leucine (CUA, CUC, CUU, and UUA) are all recogT H E D E C O D I N GO F m R N A B Y t R N A s

131

specifiesone amino acid, but most amino acidsare encoded by multiple codons(seeTable 4-1). r The AUG codon for methionine is the mosr common start codon, specifyingthe amino acid at the NH2-terminus of a protein chain. Three codons (UAA, UAG, UGA) function as stop codons and specify no amino acids. r A reading frame, the uninterrupted sequenceof codons in mRNA from a specific starr codon to a stop codon, is translated into the linear sequenceof amino acids in a polypeptide chain. r Decoding of the nucleotide sequencein mRNA into the amino acid sequenceof proteins depends on tRNAs and aminoacyl-IRNA synthetases. r All tRNAs have a similar three-dimensionalstructure that includes an acceptor arm for attachment of a specific amino acid and a stem-loopwith a three-baseanticodon sequence at its ends (seeFigure 4-20). The anticodon can base-pairwith its correspondingcodon in mRNA. r Becauseof nonstandard interactions, a tRNA may basepair with more than one mRNA codon; converselSa particular codon may base-pairwith multiple tRNAs. In each case,however,only the proper amino acid is insertedinto a g r o w i n gp o l y p e p t i d ec h a i n . Each of the 20 aminoacyl-tRNA synthetasesrecognrzesa ngle amino acid and covalently links it to a cognare

rRNA

IRNA, forming an aminoacyl-tRNA (seeFigure 4-19). This reaction activates the amino acid, so it can participate in peptide bond formation.

StepwiseSynthesis of proteins @ on Ribosomes The previous sectionshave introduced two of the major participants in protein synthesis-mRNA and aminoacylated IRNA. Here we first describethe third key player in protein synthesis-the rRNA-containing ribosome-before taking a detailed look at how all three components are brought together to carry out the biochemical eventsleading to formation of polypeptide chains on ribosomes. Similar to transcription, the complex processof translation can be divided into three stages-initiation, elongation, and terminationwhich we consider in order. \7e focus our description on translation in eukaryotic cells, but the mechanismof translation is fundamentally the same in all cells.

R i b o s o m eA s re Protein-Synthesizin Mga c h i n e s If the many components that participate in translating mRNA had to interact in free solution. the likelihood of simultaneouscollisions occurring would be so low that the rate of amino acid polymerization would be very slow. The

Proteins

.9

Subunits

Assembled ribosomes

Total: 31

23S (2900rNTs)

:.E

o o-

5S ( 1 2 0r N T s )

50s

Total: 21

70s

165 (1500 rNTs)

q,

o

t

o (J

o

J

lrj

( 1 9 0 0r N T s ) FfGURE4-22 Prokaryotic and eukaryotic ribosome components.In all cells,eachribosomeconsists of a largeand a small subunit.The two subunitscontainrRNAs(red)of differentlengths,as well as a differentset of proteins.All ribosomescontaintwo maior 132

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40s

80s

r R N Am o l e c u l e(s2 3 Sa n d 1 6 5r R N Ai n b a c t e r i a2;g Sa n d 1 g Sr R N Ai n vertebrates) and a 55 rRNA The largesubunitof verteorate ribosomesalsocontainsa 5 85 rRNAbase-paired to the 2gS rRNA The numberof ribonucleotides (rNTs)in eachrRNAtvpe is indicated

B A S t cM o L E c u L A G R E N E T tMc E C H A N t s M s

efficiencyof translation is greatly increasedby the binding of the mRNA and the individual aminoacyl-tRNAs to a ribosome. The ribosome, the most abundant RNA-protein complex in the cell, directselongationof a polypeptideat a rate of three to five amino acids added per second.Small proteins of 100-200 amino acids are thereforemade in a minute or less. On the other hand, it takes 2-3 hours to make the largest known protein, titin, which is found in muscle and contains about 30,000 amino acid residues.The cellular machine that accomplishesthis task must be preciseand persistent. With the aid of the electron microscope,ribosomeswere first discoveredas small, discrete,RNA-rich particles in cells that secretelarge amounts of protein. However, their role in protein synthesiswas not recognizeduntil reasonably pure ribosome preparations were obtained. In vitro radiolabeling experimentswith such preparationsshowed that radioactive amino acids were first incorporated into growing polypeptide chains that were associated with ribosomes before appearing in finished chains. Though there are differencesbetween the ribosomes of prokaryotes and eukaryotes,the great structural and functional similarities between ribosomes from all speciesreflects the common evolutionary origin of the most basic constituents of living cells. A ribosome is composedof three (in bacteria) or four (in eukaryotes)different rRNA molecules and as many as 83 proteins,organizedinto a largesubunit and a small subunit (Figure4-22).The ribosomal subunitsand the rRNA moleculesare commonly designatedin svedbergunits (S), a measureof the sedimentationrate of macromolecules a measure centrifuged under standard conditions----essentially, of size.The small ribosomal subunit contains a singlerRNA molecule, referred to as small rRNA. The large subunit contains a molecule of large rRNA and one molecule of 55 rRNA, plus an additional moleculeof 5.8SrRNA in vertebrates.The lengths of the rRNA molecules, the quantity of proteins in eachsubunit, and consequentlythe sizesof the subunitsdiffer between bacterial and eukaryotic cells. The assembledribosome is 70S in bacteriaand 80S in vertebrates. The sequencesof the small and large rRNAs from several thousand organismsare now known. Although the primary nucleotide sequencesof these rRNAs vary considerablS the sameparts of eachtype of rRNA theoreticallycan form basepaired stem-loops, which would generate a similar threedimensional structure for each rRNA in all organisms.The actual three-dimensionalstructuresof bacterial rRNAs from E. coli recently have been determined by x-ray crystallography of the 70S ribosome(Figure4-23).The multiple' much smaller ribosomal proteins for the most part are associated with the surfaceof the rRNAs. Although the number of protein molecules in ribosomes gready exceedsthe number of RNA molecules,RNA constitutes about 60 percent of the massof a ribosome.At the interfaceof the small and large ribosomal subunits,three local domains are formed' known as the A site, the P site, and the E site. As we'll seeshortly, these are the main sitesof interaction for the aminoacyl-tRNA and mRNA within the ribosome as protein synthesistakes place' During translation, a ribosome moves along an mRNA chain, interacting with various protein factors and tRNAs

and undergoing large conformational changes'Despite the complexity of the ribosome, great progresshas beenmade in deteimining the overall structure of bacterial ribosomesand in identifying various reactive sites' X-ray crystallographic studies on the T. thermophilus 705 ribosome, for instance, have not only revealed the dimensions and overall shape of the ribosomal subunits but also localized the positions of tRNAs bound to the ribosome during elongation of a growing protein chain. In addition, powerful chemical techniques r.r.h footprinting, which is describedin Chapter 7,have "r been used to identify specific nucleotide sequencesin rRNAs that bind to protein or another RNA. Some 40 yearc after the initial discovery of ribosomes' their overall structure and functioning during protein synthesisare finally becoming clear.

the AUG Recognizes Methionyl-tRNA;M"t Start Codon As noted earlier,the AUG codon for methionine functions as the start codon in the vast majority of mRNAs. A critical aspect of translation initiation is to begin protein synthesisat ihe start codon, thereby establishing the correct reading frame for the entire mRNA. Both prokaryotes and eukaryotes contain two different methionine tRNAs: tRNAlM"'can initiate protein synthesis, and IRNAM" can incorporate methionine only into a growing protein chain. The same aminoacyl-tRNA synthetase(MetRS) charges both tRNAs with met'hionine;however, only Met-tRNA,M" (i.e., activated methionine attached to tRNAiM't) can bind at the appropriate site on the small ribosomal subunit, the P site, to begin synthesisof a polypeptide chain' The regular Met-tRNAM"' and all other chargedtRNAs bind only to another ribosomal site, the A site, as described later. As mentioned earlier, in bacteria, the initiating methionine has a formyl group linked to its amino group, forming N-formylmethionine.

TranslationInitiation UsuallyOccursat the First A U G f r o m t h e 5 ' E n do f a n m R N A During the first stage of translation' the small and large ribosomal subunits assemblearound an mRNA that has an aminoacylated initiator IRNA correctly positioned at the start codon. This processis mediated by a specialset of proteins known as translation initiation factors (IFs). As each individual component ioins the complex, it is accompanied by one or more specific initiation factors. Interactions between theseinitiation factors help stabilizethe complex' Furthermore, some initiation factors are coupled to GTP, and the hydrolysis of GTP to GDP functions as a proofreading switch that allows subsequentsteps to proceed only if the

the two ribosomal subunits once the small subunit with a charged initiator IRNA (Met-tRNAiM"t) has bound to an ON RIBOSOMES O S T E P W I S sEY N T H E 5 I 5O F P R O T E I N S

133

Podcast:Structure of the Ribosome C) ZN Rotating 3-D Model of a BacterialRibosome < FfGURE4-23 Structureof E.coli 70Sribosomeas determined by x-ray crystallography.Modelof the ribosome viewedalongthe interface between the large(50S)andsmall(30S)subunitsThe165 rRNAandproteins in thesmallsubunitarecolored lightgreenand darkgreen,respectively; the23SrRNAandproteins in the largesubunit arecoloredlightpurpleanddarkpurple,respectively; andthe 55rRNA iscoloreddarkblue Thepositions of the ribosomal A, f andEsitesare indicatedNotethatthe ribosomal proteins arelocatedprimarily on the surface of the ribosome, andthe rRNAs on the inside, liningtheA, f andEsites[From B S Schuwirth etal.2005.Nature}l}:827 1

initiation codon in an mRNA. The first step of translation initiation is formation of a preinitiation complex. The preinitiation complex is formed when the 40S subunit complexed with the multisubunit eIF3 complex associateswith eIFIA and a ternary complex consisting of the MettRNAiM't and eIF2 bound to Ctp (Figure4-i4, steptr). The initiation factor eIF2 alrernates between association with GTP and GDP; it can bind Met-tRNA1M.. only when it is associatedwith GTP. Cells can regulate protein synthesis by phosphorylating a serine residue on the eIF2 bound to GDP; the phosphorylated complex is unable to exchange the bound GDP for GTP and therefore cannot bind MettRNAiM"t, thus inhibiting protein synthesis. The 5' cap of an mRNA to be translated is bound by the eIF4 cap-binding complex. The eIF4 cap-binding complex consists of several subunits with different functions; the eIF4E subunit of the eIF4 complex binds the 5/ cap structure on mRNAs (Figure 4-14).ThemRNA-eIF4 complix then as-

subunit so that it can remove short regions of RNA secondary structure in bound RNA using energy from ATp hydrol_ ysis. This multicomponent initiation complex then probably slides along, or scctns,the bound mRNA as the helicase activity of eIF4A unwinds RNA secondary structures that might otherwise interfere with scanningalong the mRNA in the 3' direction. Scanning srops when the tRNAiM.t anticodon recognizesthe start codon, which is the first AUG 134

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downstream from the 5' end in most eukaryotic mRNAs (step E). Recognition of the start codon leads to hydrolysis of the GTP associatedwith eIF2. an irreversible steD that prevents further scanning.Selectionof the initiating AUG is facilitated by specific surrounding nucleotides called the Kozak sequence,for Marilyn Kozak, who defined it: (5,) ACCAUGG (3'). The A precedingthe AUG (underlined)and the G immediately following it are the most important nucleotidesaffecting translation initiation efficiency.Once the small ribosomal subunit with its bound Met-tRNAiM". is correctly positioned at the start codon and the GTp bound by elF2 is hydrolyzed to GDP, elF1.,2,3, and 4 dissociate and the small subunit unites with the large (605) ribosomal subunit in a processcatalyzedby eIF5 and 6, completing formation of an 80S ribosome. Vith the entire complex assembled, the Met-tRNA1M"'bound to the AUG codon is situated in the P site. Recruitment of the large ribosomal subunit is accompaniedby hydrolysis of a GTP bound by eIF5, another proofreading step (step @). Coupling the ribosome-subunitjoining reaction to GTP hydrolysis allows the initiation process to continue only when the subunit interaction has occurred correctly. It also makes this an irreversiblestep, so that the ribosomal subunits do not dissociateuntil the entire mRNA is translated and protein synthesisis terminated. The eukaryotic protein-synthesizing machinery begins translation of most cellular mRNAs within about 100 nucleotides of the 5' capped end as just described.However, somecellular mRNAs contain an internal ribosome entry site (IRES) located far downsrream of the 5' end. In addition, translation of some viral mRNAs, which lack a 5, cap, is initiated at IRES sequencesby the host-cell machinery of infectedeukaryotic cells.Someof the sametranslation initiation factors that assistin ribosome scanningfrom a 5, cap are required for locating an internal AUG start codon, but exactly how an IRES is recognizedis lessclear.Recentresultsindicate that some IRES sequencesfold into an RNA structure that binds to the E site on the ribosome (seebelow), thereby positioning a nearby internal AUG start codon in the p site. In bacteria, binding of the small ribosomal subunit to an initiation site occurs by a different mechanism that allows initiation at internal sitesin the polycistronic mRNAs transcribed from operons.In bacterial mRNAs, an :6-base sequencecomplementary to the 3' end of the small rRNA precedesthe AUG

B A s t cM o L E c u L A G R E N E T tMc E C H A N t s M s

4-24 lnitiationof translationin eukaryotes'/nset" < FIGURE the of translation, at thetermination dissociates Whena ribosome factorselF3andelF6, with initiation associate 4OSand605subunits thatcaninitiateanotherroundof formingcomplexes respectively, of the indicated addition Sequential and Steps Z: translation Il formsthe initiation complex to the40Ssubunit-elF3 components initiation of the mRNAbythe associated complexStepB: Scanning Metbound and subunit of thesmall leadsto positioning complex subuntt large of the Association Step 4: at thestartcodon. tRNAiM"t the mRNATwoiniti(605)formsan 805ribosome readyto translate proteins, elF2(step[) andelF5(step4) areGTP-binding ationfactors, precise The initiation. duringtranslation whoseboundGTPishydrolyzed yet well not is released are factors initiation timeat whichparticular discussron detailed more lAdapted for a text the See characterized

elF6

\

\,

f r o m R M e n d e za n d J D R i c h t e t2 0 0 1 , N a t u r eR e vM o l C e l lB i o l 2 : 5 2 1 l

start codon by 4-7 nucleotides.Basepairing betweenthis sequencein the mRNA, called the Shine-Dalgarnosequenceafter iis discoverers,and the small rRNA placesthe small ribosomal subunit in the proper position for initiation. Next' f-MettRNAiM" and initiation factors comparableto eIFIA' eIF2' and eIF3 associatewith the small subunit,followed by association of the large subunit to form the complete bacterial ribosomeby a mechanismsimilar to that in eukaryotes'

Preinitiation complex AUG-

(AAA)"

e l F 4( c a p - b i n d i ncgo m p l e x )+ m R N A

wret$cre mTGppp

D u r i n gC h a i nE l o n g a t i o nE a c hI n c o m i n g Aminoacyl-tRNAMovesThroughThree R i b o s o m aSl i t e s The correctly positioned ribosome-Met-tRNAiM't complex is now ready to begin the task of stepwiseaddition of amino acidsby the in-frame translation of the mRNA' As is the case with initiation, a set of special proteins, termed translation

(AAA}"

Initiationcomplex RNA u n w i n d in q , s c a n n r n g ,a n d start srte r e c o gn r tr or l

ATP

E

ADP + P; e l F 1 A ,e l F 3 ,e l F 4c o m p l e x , + P; elF2.GDP

(AAAln 3', 6 0 5 s u b u n i t - e l F 6e,l F S ' G T P g(

codon at a time along the mRNA. At the completion of translation initiation, as noted akeady,Met-tRNA1M" is bound to the P site on the assembled SOSribosome (Figure 4-25, top). This region of the ribosome is called the P site becausethe IRNA chemically linked to the growing polypeptide chain is located here' The second aminoacyl-tRNA is brought into the ribosome as a ternary complex in associationwith EFla'GTP and becomes bound to the A site, so named becauseit is where aminoacylated tRNAs bind (step E). EFlct'GTP bound to various

+ P; e l F 6 ,e | F S ' G D P l\

80S ribosome

SN R I B O S O M E S ' S T E P W I SSEY N T H E 5 IO5 F P R O T E I NO

135

FocusAnimation:proteinSynthesis flltt

Entry of next a a - t R N Aa t A site

< FIGURE 4-25 Peptidylchainelongationin eukaryotes. Once p siteis the80Sribosome with Met-tRNA,M", in the ribosome (top),a ternary assembled complex bearing thesecond aminoacid (aar)codedbythe mRNAbindsto theA site(stepO) Following a conformational changein the ribosome induced by hydrolysis of GTp (stepZ), thelargerRNAcatalyzes in EFIcTGTP peptide bondformation betweenMetiandaa2(step!) Hydrolysis of GTpin EF2.GTp causes anotherconformational changein the ribosome that results in its translocation onecodonalongthe mRNAandshiftsthe unacylated tRNAiM"t to the EsiteandtheIRNAwiththe boundpeptrde to the p site(step4) Thecyclecanbeginagainwith bjndingof a ternary complex bearing aa3to the now openA site In thesecono and subsequent elongation cycles, theIRNAat the Esiteisejected during stepI asa resultof theconformational change induced by hydrolysis of GTPin EFIa.GTP[Adapted fromK H Nierhaus etal. 2OOO. inR A Garrett et al, eds, TheRibosome: Structure, Function, Antibiotics, andCellular lnteractions, p 319l ASMPress,

E

G T Ph y d r o l y s i s ,

L

E | \ 6.oor-r, iii,'"?il1,'""", change +

Peptidebond formation

ff*l:Ii""

136

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cHAprER 4

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l'z

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EF2.GDp*pi Jl'.--

Thus, GTP hydrolysis by EFlcr is another proofreading step that allows protein synthesisto proceed only when the correct aminoacylated IRNA is bound to the A site. This ohenomenon contributes to the fidelity of protein synthesis. \fith the initiating Met-tRNAiM" at the p site and the second aminoacyl-tRNA tightly bound at the A site,the q amino group of the secondamino acid reactswith the ,,activated" (ester-linked)methionine on rhe initiator IRNA, forming a peptidebond (Figure4-25, stepB; seealsoFigure4-17).This peptidyltransferdse reaction is catalyzed by the large rRNA, which preciselyorients the interacting atoms, permitting the reaction to proceed.The catalytic ability of the large rRNA in bacteriahas beendemonstratedby carefullyremoving the vast majority of the protein from large ribosomal subunits. The nearly pure bacterial23S rRNA can catalyzea pepridyltransferasereaction betweenanalogsof aminoacylated-tRNAand peptidyl-tRNA. Further support for the catalytic role of large rRNA in protein synthesiscomesfrom crystallographicstudies showing that no proteins lie near the site of peptide bond synthesisin the crystal structureof the bacteriallarge subunit. Following peptide bond synthesis,the ribosomeis translocated along the mRNA a distanceequal to one codon. This translocation step is monitored by hydrolysis of the GTp in eukaryotic EF2.GTP. Once translocation has occurred correctly, the bound GTP is hydrolyzed, another irreversible processthat prevents the ribosome from moving along the RNA in the wrong direction or from translocatingan incorrect number of nucleotides.As a result of conformational changesin the ribosomethat accompanyproper translocation and the resulting GTP hydrolysis by EF2, tRNA;M.t, now without its activatedmethionine,is moved to the E (exit) site on the ribosome; concurrently,the secondtRNA, now covalently bound to a dipeptide(a peptidyl-rRNA), is moved to the P site (Figure4-25, stepZf). Tianslocationthus rerurnsthe ribosome conformation to a state in which the A site is ooen and able to accept another aminoacylatedIRNA complexed with EFlcr.GTP,beginninganother cycleof chain elongation. Repetition of the elongationcycle depictedin Figure 4-25 adds amino acids one at a time to the C-terminus of the

B A s t cM o L E c u L AG R E N E T tMc E c H A N I s M s

(a)

generaldomains of all tRNAs result in the movement of the tnXRr betweenthe A, P' and E sitesas the ribosome translocatesalong the mRNA one three-nucleotidecodon at a time'

Translationls Terminatedby ReleaseFactors W h e n a S t o p C o d o nl s R e a c h e d The final stageof translation, like initiation and elongation, requireshighly specificmolecular signalsthat decidethe fate of the mRNA-ribosome-peptidyl-tRNA complex' Two (b)

eukaryotic releasefactor, eRF3, is a GTP-binding protein' The eRF3'GTP actsin concertwith eRFl to promote cleavage of the peptidyl-tRNA, thus releasingthe completed protJin chain (Figure 4-27). Bacteria have two releasefactors (RF1 and RF2) that are functionally analogousto eRFl and

A FfGURE4-26 Low-resolutionmodel of E. coli 7OSribosome. images oI E colil0S (a)Toppanels microscoprc showcryoelectron showcomputerand5OSand30SsubunitsBottompanels ribosomes in the sameorientation of images of manydozens averages derived images (b)Modelof a 7OSribosome basedon thecomputer-derived are superimposed Three tRNAs studies cross-linking andon chemical polypeptide sitesThenascent andE(yellow) P(green), on theA (pink), c h a i ni sb u r i e di n a t u n n eiln t h el a r g er i b o s o msaul b u n ti th a tb e g i n s I S Gabashvili stemof theIRNAin the Psite [See closeto the acceptor of J Frank courtesy Cell1OO:537, l et al, 2OOO, growing polypeptideas directedby the mRNA sequence,until a stop codon is encountered.In subsequentcycles,the conformational changethat occurs in step f,l electsthe unacylated IRNA from the E site. As the nascent polypeptide chain becomeslonger,it threadsthrough a channelin the large ribosomal subunit, exiting at a position opposite the side that interactswith the small subunit (Figure4-26). RNAIn the absenceof the ribosome'the three-base-pair RNA hybrid between the IRNA anticodons and the mRNA codonsin the A and P siteswould not be stable;RNA-RNA duplexesbetweenseparateRNA moleculesmust be considerably longer to be stable under physiologicalconditions. However, multiple interactions between the large and small rRNAs and general domains of tRNAs (e.g., the D and TVCG loops, seeFigure 4-20) stabilizethe tRNAs in the A and P sites,while other RNA-RNA interactionssensecorrect codon-anticodonbasepairing, assuringthat the geneticcode is read properly. Then, interactions between rRNAs and the

eRFl + eRF3.GTP

t

Peptidyl-tRNA \ cleavage | \

-+

eRFl+eRF3oGDP+Pi

A FIGURE4-27 Terminationof translationin eukaryotes'When a stopcodon(UAA' proteinchainreaches bearinga nascent a ribosome probably complex, ribosomal factoreRFlentersthe UGA,UAG),release the bound of Hydrolysis eRF3'GTP with at or neartheA sitetogether chainfromtheIRNAin of the peptide bycleavage GTPisaccompanied subunits of thetRNAsandthetwo ribosomal the Psiteandrelease ON RIBOSOMES . 5 F PROTEINS S T E P W I SS E Y N T H E S IO

137

a GTP-binding factor (RF3) that is analosousto eRF3. Once again,the eRF3 GTPasemonirors the coriect recognition of a stop codon by eRF1. The peptidyl-rRNA bond of the IRNA

We can now seethat one or more GTp-binding proteins participate in each stage of translation. These proteins belong to the GTPasesuperfamily of switch proteins that cycle between a GTP-bound active form and GDp-bound inaciive form (seeFigure 3-32). Hydrolysisof the bound GTp causes a conformational change in the GTpase itself and other associatedproteins that are critical to various complex molecular processes.In translation initiation, for instance,hydrolysis of eIF2.GTP to eIF2.GDp prevenrsfurther scanning of the mRNA once the start site is encounrered and allJws binding of the large ribosomal subunit to the small subunit (seeFigure 4-24, step p). Similarly, hydrolysis of EF2.GTp to EF2.GDP during chain elongation leads to correct translocation of the ribosome along the mRNA (seeFigure 4-25, step Zl), and hydrolysisof eRF3.GTp to eRF3.GDp assurescorrect termination of translation. Since hvdrolvsis of the high-energy B-1 phosphoesterbond of GTi, is irreversible, coupling of these stepsin protein synthesisto GTp hydrolysis preventsthem from going in rhe reversedirection. One kind of mutation that can inactivate a gene in any organism is a base-pairchange that converts a codon nor_ mally encoding an amino acid into a stop codon, e.g., UAC (encodingtyrosine) -+ UAG (stop). When this occurs early in the reading frame, the resulting truncated protein usually is nonfunctional. Such mutations are called nonsense mvta_ tions becausewhen the genetic code equating each triplet codon sequencewith a single amino *"i being dlci_ ".id phered, the three stop codons were found ,rot to .rr.od. ,.ry amino acid-they did not ,,make sense.', In geneticstudieswith the bacterium E. coli, it was dis_

the protein encoded by the original gene with the nonsense mutation is produced to provide its essentialfunctions, the effect of the first mutation is said to be suppressedby the second mutation in the anticodon of the IRNA gene. This mechanism of nonsensesuppressionis a powerful tool in geneticstudiesin bacteria.For example, mutant bacterial virusescan be isolatedthat cannot grow in normal cells, but can grow in cellsexpressinga nonsense-suppressing IRNA becausethe mutant virus has a nonsensemutation in an essential gene. Such mutant viruses grown on the nonsense-suppressingcells can then be used in experimentsto analyzethe function of the mutant geneby infecting normal cellsthat do not suppressthe mutation and analyzing what step in the viral life cycle is defectivein the absenceof the mutant protein.

Polysomesand RapidRibosomeRecycling Increasethe Efficiencyof Translation

an mRNA. Simultaneoustranslation of an mRNA bv multiole ribosomesis readily observablein electronmicrographsandty sedimentation analysis,revealing mRNA attached to multiple ribosomes bearing nascentgrowing polypeptide chains. These structures, referred to as polyribosomes or polysomes, were seen to be circular in electron micrographs of some tissues. Subsequentstudieswith yeastcellsexplained the circular shape of polyribosomes and suggestedthe mechanism by which ribosomes recycleefficiently. These studies revealed that multiple copies of a cytosolic protein found in all eukaryotic cells,poly(A)-binding prroteinI (PABPI), can interacr with both an mRNA poly(A) tail and the 4G subunit of yeast eIF4. Recall that the 4E subunit of yeast eIF4 binds to the 5' end of an nRNA. As a result of thesein-

cap. The circular pathway depictedin Figure 4-Z9b,which may operate in many eukaryotic cells, would enhanceribosome re_ cycling and thus increasethe efficiency of protein synthesis.

Stepwise Synthesisof proteins on Ribosomes prokaryoric and eukaryotic ribosomes-the large .Both bonucleoproteincomplexeson which translation o..urrconsistof a small and a large subunit (seeFigure4-22).Each subunit contains numerous different proteins and one major rRNA molecule (small or large). The large subunit also contains one accessory55 rRNA in bacteriaand two acces, sory rRNAs in eukaryores(5S and 5.8S in vertebrates). 138

.

c H A p r E4R | B A S tM c oLEcuLA GRE N E TM t cE c H A N t s M s

ing GTP-binding proteins that hydrolyze their bound GTP TJGDP whe.t aitip has been completed successfully' During initiation' the ribosomal subunits assemblenear e translation start site in an mRNA molecule with the IRNA carrying the amino-terminal methionine (MettRNAiM"t) base-pairedwith the start codon (Figute 4-24)' ain elongation entails a repetitive four-step cycle: (1) binding of an incoming aminoacyl-tRNA to the A site e ribosome, (2) tight binding of the correct aminoacylIRNA to the A site accompanied by releaseof the previ-

to the E site (seeFigure4-25).

(b)

E'

leads to translocation. r Termination of translation is carried out by two types of termination factors: those that recognizestop codons and those that promote hydrolysis of peptidyl-tRNA (see Figure 4-27). Once again, correct recognition of a stop codon is monitored by a GTPase(eRF3)' The efficiency of protein synthesis is increased by the siultaneoustranslation of a singlemRNA by multiple ribo-

4-28 The circularstructureof mRNA FIGURE a EXPERIMENTAL mRNAformsa circular increasestranslationefficiency.Eukaryotic of threeproteins(a)In the presence owingto interactions structure eukaryotic protein| (PABPI), andelF4G, elF4E, of purifiedpoly(A)-binding visiblein thisatomicforcemicrograph structures, formcircular mRNAs interactlons protein-protein andprotein-mRNA Inthesestructures, the 5' and3' endsof the mRNA.(b)Model forma bridgebetween of ribosomal polysomes andrecycling on circular of proteinsynthesis translate cansimultaneously ribosomes Multipleindividual subunits. by formstabilized mRNA,shownherein circular a eukaryotic boundat the 3' and5' ends.Whena betweenproteins interactions fromthe 3' end,the anddissociates translation completes ribosome and 5' cap(m7G) nearby find the can rapidly subunits separated of A Sachs ] [Part(a)courtesy initrateanotherroundof svnthesrs. r Analogous rRNAs from many different speciesfold into quite similar three-dimensionalstructures containing numerous stem-loopsand binding sites for proteins, mRNA, and tRNAs. Much smaller ribosomal proteins are associated with the periphery of the rRNAs. r Of the two methionine tRNAs found in all cells,only one (tRNAiM't) functions in initiation of translation. r Each stage of translation-initiation, chain elongation, and termination-requires specific protein factors, includ-

!f,l

DNAReplication

Now that we haveseenhow geneticinformationencodedin

(duplex\DNA strandswould form a new double-stranded intact' In a remain molecule,and the parentalduplexwould are perstrands parental mechanism,the semiconservative with molecule a duplex and eachforms manentlyseparated, evidence Definitive it. to the daughterstrand base-paired DNA REPLICATION

139

(a) Predicted results C o n s e r v a t i vm e echanism

(b) Actual results

S e m i c o n s e r v a t i vmee c h a n r s m

Density-

Density-

Generation 0

P a r e n t asl t r a n d s s y n t h e s i z e idn 1 5 N

HH

0.7

HH

1.0 1.1 After first d o u b l i n gi n t a N

1.5 1.9 HH

/\

A

LL

/\

/\

LH

2.5

/\

3.0 4.1

After second d o u b l i n gi n t r N

0 and 1.9 mrxed 0 and4.1 mrxed HHLL

LLLL

LLHL

IGURE4-29 The Meselson_Stahl experiment be semiconservative.Thrsexperimentshowed a semiconseryative mechanism. E colicells initially weregrown in a mediumcontaining ammoniumsaltsprepared wlth "heavy"nitrogen(1sN)untilallthe cellularDNAwas labeledAfter the cellsweretransferred to a mediumcontaining the normal,,light,, isotope(raN),samples were removedperiodically from the cultures and the DNA in eachsamplewas analyzed by equilibrium density_gradient centrifugation, a procedure that separates macromolecules on the basis of theirdensity. Thistechniquecan separate (H_H), heavy_heavy light_ light(L-L),and heavy-light (H-L)duplexes into distinctbands. (a) Exp_ected compositionof daughterduplexmoleculessynthesized from 1sN-labeled DNAafterF. colicellsareshiftedto raN_containing medium if DNA replicationoccursby a conservative or semtconseryative mechanismParental heavy(H)strandsarein red;light(L)strands synthesized aftershiftto laN-containing mediumarein brue.Notethat the conservative mechanism nevergenerates H_LDNAand that the semiconservative mechanism nevergenerates H_HDNA but does generateH-LDNA duringthe secondand subsequent doublingsWith additionalreplication cycles, the lsN-labeled (H)strandsfrom the originalDNAarediluted,so that the vastbulkof the DNAwould consist that duplex DNA is replicated by a semiconservarive mech_

LHLL

L-L H-L H-H L-L H-L H-H of L-Lduplexes with eithermechanism(b)Actualbandingpatternsof DNAsubjected to equilibrium density-gradient centrifugationbefore and aftershiftinglsN-labeled E colicellsto laN-containing medium. DNA bandswerevisualized underUV lightand photographedThe traceson the left are a measureof the densityof the photographic signal,and hencethe DNAconcentratlon, alongthe lengthof the centrifugecellsfrom left to right.The numberof generations(far left) followingthe shiftto 1aN-containing mediumwas determined by countrngthe concentration of E colicellsin the cultureThisvalue corresponds to the numberof DNA replicatron cyclesthat had occurred at the time eachsamplewas taken.After one generation of growth,all the extractedDNA had the densityof H-LDNA.After '19 generations, approximately halfthe DNA hadthe densityof H-LDNA;the otherhalf had the densityof L-LDNA.With additionalgenerations, a largerand largerfractionof the extractedDNA consistedof L-Lduplexes;H_H duplexesneverappearedTheseresultsmatchthe predictedpatternfor the semiconservative replicationmechanismdepictedin (a) The bottom two centrifuge cellscontainedmixtures of H-HDNAand DNA isolated at 1 9 and 4 1 generations in orderto clearlyshowthe positions of H_H, H-1,and L-LDNA in the densitygradient.[part(b)fromM Meselson andF. W Stahl, 1958,Proc Nat'lAcadSciUSAM:6711

DNA strands. However, the vast preponderance of RNA and DNA in cells is synthesized from preexisting duplex DNA.

D N A P o l y m e r a s eRs e q u i r ea p r i m e r t o I n i t i a t eR e p l i c a t i o n Analogousto RNA, DNA is synthesized from deoxynucleoside 5'-triphosphateprecursors(dNTps). Also like RNA synthesis, DNA synthesisalwaysproceedsin the 5,+3, direction because chain growth resultsfrom formation of a phosphoesterbond 140

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cHAprER 4

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beftveenthe 3' oxygen of a growing strand and the o phosphate of a dNTP (seeFigure 4-10a). As discussedearlier, an RNA polymerasecan find an appropriate transcription start site on duplex DNA and initiate the synthesisof an RNA complementaryto the template DNA strand (seeFigure 4-11). In contrast, DNA polymerasescannot initiate chain synthesisde novo; instead, they require a short, preexisting RNA or DNA strand, called a primer, to begin chain growth. \7ith a primer base-pairedto the template strand, a DNA polymeraseadds deoxynucleotidesto the free hydroxyl group at the 3' end of the primer as directed by the sequenceof the template strand: Primer

lVhen RNA is the primer, the daughter strand that is formed is RNA at the 5' end and DNA at the 3' end.

D u p l e xD N A l s U n w o u n da n d D a u g h t e rS t r a n d s A r e F o r m e da t t h e D N A R e p l i c a t i o nF o r k In order for duplex DNA to function as a template during replication,the two intertwined strandsmust be unwound' or melted, to make the basesavailablefor basepairing with the basesof the dNTPs that are polymerizedinto the newly synthesized daughter strands. This unwinding of the parental DNA strandsis by specifichelicases,beginningat unique segments in a DNA moleculecalledreplication origins' or simply origins. The nucleotide sequencesof origins from different organisms vary gready,although they usually contain A'Trich sequences.Once helicaseshave unwound the parental DNA at an origin, a specializedRNA polymerasecalled primase forms a short RNA primer complementaryto the unwound template strands. The primer, still base-pairedto its complementary DNA strand, is then elongated by a DNA polymerase,thereby forming a new daughter strand.

cent fragments'

SeveralProteinsParticipatein DNA Replication Detailed understanding of the eukaryotic proteins that participate in DNA replication has come largely from studies witlh small viral DNAs, particularly SV40 DNA, the circular

llil+ FocusAnimation:NucleotidePolymerization by DNA Polyrngra:g DNA 4-30 leading-strandand lagging-strand > FIGURE to each areaddedby a DNApolymerase Nucleotides synthesis. (indicated by growingdaughter strandin the 5'-+3' direction f roma continuously strandissynthesized Theleading arrowheads) (red) is strand lagging end. The primer at its 5' singleRNA thatare frommultipleRNAprimers discontinuously synthesized duplexis aseachnewregionof the parental formedperiodically produces Okazaki initially of theseprimers unwoundElongation prevtous the growing approaches fragment As each fragments, areligated andthefragments primer, the primerisremoved of the entire in synthesis results eventually of thisprocess Repetition l a g g i nsgt r a n d .

5', P o i n to f j o i n i n g L a g g i n gs t r a n d Okazakifragment P a r e n t aD l NAduPlex

S h o r tR N A p r i m e r

5', Leadingstrand

DNA REPLICATION

14'l

FocusAnimation:Coordinationof Leading- and

flltt strandsynthesis

(a) SV40DNA replicationfork

E::i1,""';

31

5',

Primase

L a g g i n gs t r a n d

E

pPol E Rfc PCNA

Primer

(b}PCNA RPA

a

Doublestranded DNA

Leadingstrand

(c)RPA

FIGURE 4-31 Model of an SV40DNA replicationfork. (a)A hexamer of largeT-antigen ([), a viralprotein. functions asa helicase to unwindthe parental DNAstrands. Single-strand regions of the parental template unwoundby largeT-antigen areboundby multiple copies of the heterotrimeric proteinRpA(Z). Theleading strandissynthesized by a complex of DNApolymerase S (polS),

(E). (b)Thethreesubunits fragment of pCNA,shownin different colors, forma circular structure with a centralholethroughwhich double-stranded DNApasses. A diagram of DNAisshownjn the centerof a ribbonmodelof the pCNAtrimer.Thediaqram at the

pCNAboundto DNAin upperleftshowsthe iconrepresenting parta. (c)Thelargesubunitof RPAcontains two domains thatbind single-stranded DNA.Onthe left,thestructure determined for the two DNA-binding domains of the largesubunitboundto single_ stranded DNAisshownwith the DNAbackbone (whitebackbone with bluebases) parallel to the planeof the page Notethatthe singleDNAstrandisextended withthe bases exposed, an optimal conformation for replication by a DNApolymerase Onthe right,the viewisdownthe lengthof thesingleDNAstrand,revealing how RpA wraparoundthe DNA.Thediagram B strands at bottomcenter showsthe iconrepresenting heterotrimeric RpAboundto singlestranded DNAin part(a).[part (a)adapted fromS J Flint etal, 2000, pathogenesis, Virology: Molecular Biology, andControl, ASMpress; part(b) afterJ M Gulbis et al, 1996,Cetl87:297; andpart(c)afterA Bochkarev etal, 1997, Nature 385:176 l

Figure 4-31 depicts the multiple proteins that coordinate copying of SV40 DNA at a replication fork. The assembled proteins at a replication fork further illustrate the concept of molecular machines introduced in Chapter 3. These multi_ component complexes permit the cell to carry out an ordered sequenceof eventsthat accomplishessentialcell functions. The molecular machine that replicatesSV40 DNA con_ tains only one viral protein. All other proteins involved in 142

o

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I

B A s t cM o L E c u L A R G E N E T tM c ECHANtsMs

llll+ FocusAnimation:Bidirectional of DNA Reptication EcoRl

SV40 DNA replication are provided by the host cell. This viral protein, large T-antigen, forms a hexamer that unwinds the parental strandsat a replication fork. Primers for leading and lagging daughter-strandDNA are synthesizedby a complex of primase, which synthesizesa short RNA primer, and DNA polymerased (Pol a), which extendsthe RNA primer with deoxynucleotides,forming a mixed RNA-DNA primer' The primer is extended into daughter-strand DNA by DNA polymerase6 (Pol 6), which is lesslikely to make errors during copying of the templatestrand than is Pol ct becauseof its proofreading mechanism (see Section 4.6 below). Pol E forms a complex with R/c (replication factor C) and PCNA (proliferating cell ntclear antigen), which displaces the primase-Pol crcomplex following primer synthesis.As illustrated in Figure 4-31,b,PCNA is a homotrimeric protein that has a central hole through which the daughterduplex DNA passes, thereby preventing the PCNA-Rfc-Pol 6 complex from dissociating from the template.Pol E is the main polymeraseused by eukaryotes for elongating DNA strands during replication. After parental DNA is separatedinto single-strandedtemplatesat the replication fork, it is bound by multiple copiesof RPA (replicationprotein A), a heterotrimericprotein (Figure 4-31,c).Binding of RPA maintains the template in a uniform conformation optimal for copying by DNA polymerases. Bound RPA proteins are dislodgedfrom the parental strands by Pol cr and Pol 6 as they synthesizethe complementary strandsbase-pairedwith the parental strands. Severaleukaryotic proteins that function in DNA replication are not depictedin Figure 4-31. DNA polymerasee also contributes to the synthesisof cellular chromosomal DNA, though its exact role is uncertain. Still other specializedDNA polymerasesare involved in repair of mismatchesand damagedlesionsin DNA (seeSection4.6). A topoisomeraseassociateswith the parental DNA aheadof the helicaseto remove torsional stressintroduced by the unwinding of the parental strands. Ribonuclease H and FEN I remove the ribonucleotidesat the 5' ends of Okazaki fragments;these are replacedby deoxynucleotidesadded by DNA polymerase6 as it Okazaki extendsthe upstream Okazaki fragment. Successive fragments are coupled by DNA ligase through standard 5'-+3' phosphoesterbonds.Replicationof a linear DNA molecule presentsa specialproblem at the ends of the molecule sincethe 5'-most RNA primers of the lagging strandscannot be replacedby DNA by this mechanism.In most eukaryotes' this problem is solved by the RNA-protein complex called telomerasethat carriesits own templateas discussedin Chapter 6, Genes,Genomics,and Chromosomes.

D N A R e p l i c a t i o nU s u a l l yO c c u r sB i d i r e c t i o n a l l y f r o m E a c hO r i g i n As indicatedin Figures4-30 and 4-31, both parental DNA strands that are exposedby local unwinding at a replication fork are copied into a daughter strand. In theory, DNA replication from a singleorigin could involve one replication fork

C i r c u l a vr i r a l cnromosome

o

q)

o)

E

.,*.,."0, o g;-ri.r-" o";"; r;,;*; a EXPERTME;I of microscopy bidirectionalreplicationof SV40DNA.Electron growthof DNAstrands bidirectional SV40DNAindicates replicating cells viralDNAfromSV4O-infected froman origin.Thereplicating one site recognizes which EcoRl, enzyme restriction wascut by the for a a landmark DNA.Thiswasdoneto provide in thecircular recognition the FcoRl in the 5V40genome: sequence specific asthe endsof linearDNA recognized isnoweasily sequence micrographs Electron microscopy. by electron vrsualized molecules a collection showed molecules DNA SV40 replicating of EcoRl-cut "bubbles," longerreplication with increasingly of cut molecules from eachendof the cut whosecentersarea constantdistance with Thisfindingisconsistent chaingrowthin two molecules at thecenterof a bubble, froma commonoriginlocated directions etal, G C Fareed diagrams' [See in thecorresponding asillustrated of N P Salzman l photographs courtesy J Virol10:484; 1972, that moves in one direction. Alternatively, two replication forks might assembleat a single origin and then move in opposite directions, leading to bidirectional growth of both i",rght., strands.Severaltypes of experiments,including the orr. sho*tt in Figure 4-32, provided early evidencein support of bidirectional strand growth. The general consensusis that all prokaryotic and eukaryoticlels employ a bidirectional mechanism of DNA DNA REpL1CAT;ON o

143

FocusAnimation:Coordination of Leading-and Lagging-strand flltt Synthesis Helicases

< FIGURE 4-33 Bidirectional mechanism of DNAreplication. Theleftreplication fork hereiscomparable to the replication fork diagrammed in Figure 4-31, whichalsoshowsproteins otherthan largeT-antigen lop:TwolargeT-antigen hexameric helicases first EJrn*'no'nn bindat the replication originin opposite orientations. Step[: Using energyprovided fromATPhydrolysis, the helicases movein opposite directions, unwinding the parental DNAandgenerating single-strand templates thatareboundby RPAproteinsStepE: primase_pol o complexes synthesize (red)base-paired shortprimers to eachof the parental primersynthesis separated strands. Stepg: pCNA-Rfc-pol 6 complexes E t".o,nn-.trand replace the primase-Pol crcomplexes J andextendtheshortprimers, generating (darkgreen)at eachreplication the leading strands fork Step@: Thehelicases furtherunwindthe parental strands, andRpA proteins bindto the newlyexposed single-strand regions. Stepg: I PCNA-Rfc-Pol 6 complexes extend the leading strands further Step6: extension ! | Leading-strand Primase-Pol ctcomplexes primers synthesize for lagging-strand t synthesis at eachreplication fork Stepfl: pCNA-Rfc-pol 6 complexes displace the primase-Pol o complexes andextendthe lagging-strand (lightgreen), Okazaki fragments whicheventually areligatedto the 5' endsof the leading strandsTheposition whereligation occursis represented by a circleReplication continues byfurtherunwinding of theparental strands andsynthesis of leading andlagging strands asin Steps4-Z Althoughdepicted asindividual stepsfor clarity, unwinding andsynthesis of leading andlaggingstrands occurconcunentlv I

Unlike SV40 DNA, eukaryotic chromosomal DNA moleculescontain multiple replication origins separatedby tens to hundreds of kilobases.A six-subunit protein called ORC, for origin recognition complex, binds to each origin and associateswith other proteins required to load cellular hexameric helicasescomposed of six homologous MCM proteins (for minichromosome maintenance. the genetic screen initially_used to identify the genes encoding them). Two opt.nn,"n-strand primersynrhesis posed MCM helicases separatethe parental strands at an

extension El I Leaoing-strand

+

E J

extension Z I a.nn,nn-strand

+

transcription of most genes,control of the initiation step is the primary mechanismfor regulatingcellular DNA replication. Ac_ S t r a n dl i g a t i o n

replication. In the caseof SV40 DNA, replication is initiated by binding of two large T-antigen hexamiric helicasesto the

into two daughtercells.We discussthe various regulatory mech_ anismsthat determinethe rate of cell division in Chapter 20.

DNA Replication Each strand in a parental duplex DNA acts as a template r synthesisof a daughter strand and remains base-paired the new strand, forming a daughter duplex (using a 144

C H A P T E R4

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B A S T CM O L E C U L A RG E N E T T M C ECHANTSMS

semiconservativemechanism).New strands are formed the 5'--+3'direction. r Replication begins at a sequencecalled an origin. Each eukaryotic chromosomal DNA molecule contains multiple replication origins. r DNA polymerases,unlike RNA polymerases,cannot unwind the strandsof duplex DNA and cannot initiate synthesis of new strandscomplementaryto the templatestrands. r At a replication fork, one daughter strand (the leading strand) is elongated continuously. The other daughter strand (the lagging strand) is formed as a seriesof discontinuous Okazaki fragments from primers synthesizedevery few hundred nucleotides(Figure 4-30). r The ribonucleotidesat the 5' end of each Okazaki fragment are removed and replacedby elongation of the 3' end of the next Okazaki fragment. Finally adjacent Okazaki fragments are joined by DNA ligase. r Helicasesuse energyfrom ATP hydrolysis to separatethe parental (template)DNA strands. Primase synthesizesa short RNA primer, which remains base-pairedto the template DNA. This initially is extendedat the 3' end by DNA polymerase a (Pol c), resulting in a short (S')RNA(3' )DNA daughterstrand. r Most of the DNA in eukaryotic cells is synthesizedby Pol 6, which takes over from Pol ct and continues elongation of the daughterstrandin the 5'+3' direction.Pol 6 remains stably associatedwith the template by binding to Rfc protein, which in turn binds to PCNA, a trimeric protein that encirclesthe daughter duplex DNA (seeFigure 4-31). r DNA replication generally occurs by a bidirectional mechanism in which two replication forks form at an origin and move in opposite directions, with both template strandsbeing copied at eachfork (seeFigure4-33)' r Synthesisof eukaryotic DNA in vivo is regulatedby controlling the activity of the MCM helicasesthat initiate DNA replication at multiple origins spacedalong chromosomal DNA.

![

DNARepairand Recombination

Damage to DNA is unavoidable and arises in many ways. DNA damage can be caused by spontaneouscleavageof chemicalbonds in DNA, by environmental agentssuch as ultraviolet and ionizing radiation, and by reaction with genotoxic chemicalsthat are by-products of normal cellular metabolism or occur in the environment' A mutation in the normal DNA sequencecan occur during replication when a DNA polymerase inserts the wrong nucleotide as it reads a damagedtemplate. Mutations also occur at a low frequency as the result of copying errors introduced by DNA polymeraseswhen they replicate an undamagedtemplate. If such mutations were left uncorrected, cells might accumulate so many mutations that they could no longer function properly, In addition, the DNA in germ cells might incur too many

mutations for viable offspring to be formed' Thus the prevention of DNA sequenceerrors in all types of cellsis important for survival, and several cellular mechanismsfor repairing damagedDNA and correcting sequenceerrors have evolved' One mechanismfor repairing double-strandedDNA breaks' by a processcalled recombination, is also used by eukaryotic cells to generatenew combinations of maternal and paternal geneson each chromosome through the exchangeof segments of the chromosomesduring the production of germ cells (e'g', sperm and eggs). Significantln defects in DNA repair mechanisms and cancerare closelyrelated. !7hen repair mechanismsare compromised, mutations accumulate in the cell's DNA. If these mutations affect genes that are normally involved in the careful regulation of cell division, cells can begin to divide uncontrollably, leading to tumor formation, and cancer' Chapter 25 outlines in detail how cancer arisesfrom defects in DNA repair.We will encountera few examplesin this section, as well, as we first consider the ways in which DNA integrity can be compromised' and then discussthe repair mechanismsthat cells have evolved to ensurethe fidelity of this very important molecule'

IntroduceCopyingErrors DNA Polymerases and Also CorrectThem The first line of defensein preventingmutations is DNA polymerase itself. Occasionally, when replicative DNA polymerasesprogressalong the template DNA, an incorrect nuto the growing 3' end of the daughterstrand cleotideit for instance,in(seeFigure"da.a 4-31,).E. coll DNA polymerases., troduce about 1 incorrect nucleotideper 104 polymerizednucleotides.Yet the measuredmutation rate in bacterial cells is much lower: about 1 mistake in 10e nucleotidesincorporated into a growing strand. This remarkable accuracy is largely due to proofreading by E. coli DNA polymerases' Pro-ofreadingdependson a 3'-+5' exonucleaseactiuity of some DNA polymerases.Sfhen an incorrect baseis incorporated during DNA synthesis,base-pairingbetweenthe 3' nucleotideof the nascentstrand and the templatestrand doesnot occur.As a result,the polymerasepauses'then transfersthe 3' end of the growing chain to its exonucleasesite, where the incorrectmispairedbaseis removed (Figure4-34)' Then the 3' end is transferredback to the polymerasesite' where this region is copied correctly. Like the E. coli DNA polymerases' I*o enkaryotic DNA polymerases,6 and e, used for replication of most chromosomal DNA in animal cells' also have proofreading activity. It seemslikely that proofreading is indispensablefor all cellsto avoid excessivemutatlons'

C h e m i c aal n d R a d i a t i o nD a m a g et o D N A Can Leadto Mutations DNA is continually subiectedto a baffage of damaging chemical reactions; estimatesof the number of DNA damin a singlehuman cell range from 10a to 105 per "g..,r..t,, day! Even if DNA were not exposedto damaging chemicals, ..it"in aspectsof DNA structure are inherently unstable' D N A R E P A I RA N D R E C O M B I N A T I O N O

145

Frngers

Thumb

t^

Thumb

Fingers urowtng strand

Pol

Template strand

Exo

FIGURE 4-34 Proofreading by DNApolymerase. All DNA polymerases havea similar three-dimensional structure, which resembles a half-opened righthandThe,,fingers,, bindthesinglestranded segment of thetemplatestrand,andthe polymerase catalytic (Pol)liesin thejunctionbetween activity thefingers andpalm.As long asthecorrect nucleotides areaddedto the3, endof thegrowing strand,it remains in the polymerase site Incorporation of an incorrect

baseat the3' endcauses melting of thenewlyformedendof the duplexAsa result, pauses, thepolymerase andthe3,endof the growingstrandistransferred to the 3,-+5,exonuclease site(Exo) about3 nm away,wherethe mtspaired baseandprobably otherbases areremoved. Subsequently, the 3' endflipsbackintothe polymerase siteandelongation resumes. fromC M Joyce andT.T.Steltz, [Adapted 1995, 1 Bacteriol 177:6321,and S Bellandl Baker, 1998, Ceil92:2951

For example, the bond connecting a purine baseto deoxyribose is prone ro hydrolysis at low rate under physiological conditions, leaving a sugar without an attached base.Thus coding information is lost, and this can lead to a mutarion during DNA replication. Normal cellular reacrions,including the movement of electronsalong the electron-transportchain in mitochondria and lipid oxidation in peroxisomei, produce severalchemicalsthat react with and damage DNA, including hydroxyl radicals and superoxide (O2 ). These too can causemutations, including those that lead to cancers. Many spontaneous mutations are point mutations, which involve a changein a single basepair in the DNA se-

quence. One of the most frequent point mutations comes ftom deamination of a cytosine (C) base,which converts it into a uracil (U) base. In addition, the common modified baseS-methylcytosineforms thymine when it is deaminated. If thesealterationsare not correctedbefore the DNA is replicated,the cell will usethe strandcontainingU or T as template to form a U.A or T.A basepair, thus creating a permanent changeto the DNA sequence(Figure 4-35). Radiation from the environment can also have dramatic consequences for DNA. High-energy ionizing radiation such as x-rays and gamma rays causedouble-strandedbreaks in DNA. Uy radiation found in sunlight causesdistortions in

NH.

U

l' N'c-c-cr. ttl ozc'-r-cH I

Deamination

(.tl HNz"\c-cH"

trl

o-rctN.c

2-Deoxyribose

2-Deoxyribose

5-Methylcytosine

Thymine

5',

?21

Deamination

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5',

J

Replication

z

E .* Base-excision repatr 3',

3',

Witd-type DNA FIGURE 4-35 Deamination leadsto point mutations.A spontaneous pointmutation canformbydeamination of 5-methylcytosine (C)to formthymine(T).lf the resulting T.Gbasepair isnot restored to the normalC.Gbasepairby baseexcision_repair 146

.

Mutant DNA

5',

Wild-type DNA

(step[), it willleadto a permanent mechanisms changeIn sequence (r.e., a mutation) following (stepZ). Afteroneround DNAreplication of replication, onedaughter DNAmolecule willhavethe mutantT.A basepairandtheotherwillhavethewild-type C.Gbasepair.

c H A p r E4R | B A s t cM o L E c u L AGRE N E TM t cE c H A N t s M s

the DNA double helix that interfere with proper replication and transcription.

High-FidelitD y N A E x c i s i o n - R e p aSi ry s t e m s a n d R e p a i rD a m a g e Recognize In addition to proofreading,cellshaveother repair systemsfor preventingmutations due to copying errors, spontaneousmutation, and exposureto chemicalsand radiation. SeveralDNA excision-repair systemsthat normally operate with a high degree of accvracy have been well studied. These systemswere first elucidated through a combination of genetic and biochemicalstudiesin E. coli. Homologs of the key bacterialproteins exist in eukaryotes from yeast to humans, indicating that theseerror-freemechanismsaroseearly in evolution to protect DNA integrity. Each of these systems functions in a similar manner-a segmentof the damagedDNA strand is excised, and the gap is filled by DNA polymeraseand ligaseusing the complementary DNA strand as template. I7e will now turn to a closer look at some of the mechanisms of DNA repair, ranging from repair of single basemutations to repair of DNA broken acrossboth strands. Some of theseaccomplish their repairs with great accuracy;others are lessprecise.

BaseExcisionRepairsT.G Mismatchesand DamagedBases In humans, the most common type of point mutation is a C to T, which is causedby deamination of S-methyl C to T (see Figure 4-35). The conceptual problem with base excision repair in this case is determining which is the normal and which is the mutant DNA strand, and repairing the latter so that it is properly base-pairedwith the normal strand. Sincea G.T mismatch is almost invariably causedby chemical conversion of C to U or S-methyl C to T, the repair system evolvedto removethe T and replaceit with a C (Figure4-36). The G'T mismatch is recognized by a DNA glycosylase that flips the thymine base out of the helix and then hydrolyzes the bond that connects it to the sugar-phosphate DNA backbone. Following this initial incision, an apurinic (AP) endonucleasecuts the DNA strand near the abasicsite. The deoxyribose phosphate lacking the base is then removed and replaced with a C by a specializedrepair DNA polymerasethat reads the G in the template strand. As mentioned earlier, this repair must take place prior to DNA replication becausethe incorrect base in this pair, T, occurs naturally in normal DNA. Consequently,it would be able to engagein normal \Tatson-Crick basepairing during replication, generatinga stablepoint mutation that is now unable to be recognizedby repair mechanisms(seeFigure 4-35, s t e pZ ) . Human cells contain a battery of glycosylases'each of which is specific for a different set of chemically modified DNA bases.For example,one removesS-oxyguanine,an oxidized form of guanine, allowing its replacementby an undamagedG, and others remove basesmodified by alkylating agents.The resultingnucleotidelacking a baseis then replaced

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4-36 Baseexcisionrepair of a T'G mismatch.A DNA A FIGURE formedby usually for G'Tmismatches, glycosylase specific (see flipsthethymine 4-35), Figure residues of 5-mC deamination baseout of the helixandthencutsit awayfromthesugar-phosphate (blackdot).An justthe deoxyribose (stepn), leaving DNAbackbone site[apurinic baseless icfor the resultant specif endonuclease (stepE), and thencutsthe DNAbackbone | (APE1)l endonuclease apurinic endonuclease, by an phosphate is removed thedeoxyribose with DNApolymerase associated B, a specialized lyase(APlyase), usedin repair(stepB). Thegapisthenfilledin by DNApolymerase (stepZl),restoring theoriginal by DNAligase DNAPolB andsealed 42:2946] Chemie Angewandte pair 2003, O Schdrer, G.Cbase [After

by the repair mechanismjust discussed.A similar mechanism functions in the repair of lesions resulting from depwrination, the loss of a guanine or adenine base from DNA resulting from hydrolysis of the glycosylic bond between deoxyribose and the base.Depurination occursspontaneouslyand is fairly common in mammals. The resulting abasicsites,if left unrepaired, generatemutations during DNA replication because they cannot specifythe appropriatepaired base.

MismatchExcisionRepairsOther Mismatches a n d S m a l lI n s e r t i o n sa n d D e l e t i o n s Another process,also conservedfrom bacteria to man' principally eliminates base-pair mismatches and insertions or deletionsof one or a few nucleotidesthat are accidentallyintroduced by DNA polymerasesduring replication. As with base excision repair of a T in a T'G mismatch, the conceptual problem with mismatch excision repair is determining whic^his the normal and which is the mutant DNA strand, and repairing the latter. How this happensin human cellsis not known with certainty. It is thought that the proteins that bind to the mismatchedsegmentof DNA distinguishthe template AND RECOMBINATION . DNA REPAIR

147

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Figure 4-39 illustrates how the nucleotide excisionrepair system repairs damaged DNA. Some 30 proteins are involved in this repair process,the first of which were identified through a study of the defectsin DNA repair in cultured cellsfrom individuals with xeroderma pigmentosum,a hereditarydiseaseassociatedwith a predisposition to cancer.Individuals with this diseasefrequently develop the skin cancers called melanomas and squdmous cell carcinomasif their skin is exposedto the UV rays in sunlight. Cells of affected patients lack a functional nucleotide excision-repair system. Mutations in any of at least seven different genes, called XP-A through Xp-G,

FIGURE 4-37 Mismatchexcisionrepairin humancells.The mismatch excision-repair pathway corrects errorsintroduced during replication A complex of the MSH2andMSH6proteins (bacterial MutShomologs 2 and6) bindsto a mispaired segment of DNAin sucha wayasto distinguish between thetemplate anonewty synthesized (steptr) Thistriggers daughter strands bindingof MLH.j andPMS2(bothhomologs of bacterial MutL)Theresulting DNAproteincomplex thenbindsan endonuclease thatcutsthe newly synthesized daughter strandNexta DNAhelicase unwinds the helix, andan exonuclease removes several nucleotides fromthecutendof the daughter strand,including the mismatched base(stepf,l) Finally, aswith baseexcision repair, thegapisthenfilledin by a DNA (Pol6, in thiscase) polymerase andsealed (stepS) by DNAligase

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and daughter strands; then the mispaired segmentof the daughter strand-the one with the replication error-is excisedand repaired to becomean exact;omplement of the templatestrand (Figure4-37).In contrastto baseexcisionrepair, mismatch excision repair occurs after DNA replication.

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Predispositionto a colon cancerknown as bereditary nonpolyposiscolorectalcancerresultsfrom an inherited loss-of-function mutation in one copy of either the MLHL or the MSH2 gene. The MSH2 and MLH1 proteins are essentialfor DNA mismatch repair (seeFigure 4-37). Cells with ar least one functional copy of each of these

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mon in noninherited forms of colon cancer.I 148

CHAPTER 4

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FIGURE4-38 Formation of thymine-thymine dimers. The most commontype of DNA damagecausedby UV irradiation, thymine-thymine drmerscan be repairedby an excision-repair mechanism

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vast majority of women with inherited susceptibility to breast cancer have a mutation in one allele of either the rttttttl BRCA-1 or the BRCA-2 genesrhat encodeproteins participating in this repair process.Loss or inactivation of the second allele inhibits the homologous recombination repair pathway and thus tends to induce cancerin mammary or ovarian epithelialcells,although at presentit is not clear why these estrogen-responsive tissuesare favored sites of carcinogenesis. Yeastscan repair double-strandbreaks induced by 1-irradiation. Isolation and analysisof radiationsensitive(RAD) mutants that are deficient in this reoair system facilitated study of the process. Virtually all the proteins a yeast Rad proteins have homologs in the human genome, Jo,n". and the human and yeastproteins function in an essentially identical fashion. A variety of DNA lesions not repaired by mechanisms discussedearlier can be repaired by mechanismsin which the damagedsequenceis replaced by a segmentcopied from the same or a highly homologous DNA sequenceon the homologous chromosome of diploid organisms,or the sister chromosome following DNA replication in all organisms.These mechanismsinvolve an exchange of strands between separate DNA moleculesand henceare collectivelyreferred to as FIGURE 4-40 Nonhomologous end-joining.Whensister D N A r ecombination mechanisms. chromatids arenot available to helprepairdouble-strand breaks, In addition to providing a mechanism for DNA repair, nucleotide sequences arebuttedtogetherthatwerenot apposed in similar recombination mechanismsgenerategeneticdiversity the unbroken DNA TheseDNAendsareusually fromthesame among the individuals of a speciesby causing the exchange chromosome locus,andwhenlinkedtogether, several basepairsare of large regions of chromosomesbetween the maternal and lost Occasionally, endsfromdifferent chromosomes areaccidentally paternal pair of homologous chromosomesduring the spejoinedtogetherA complex of two proteins, KuandDNA-dependent cial type of cellular division that generatesgerm cells (sperm protern kinase, bindsto theendsof a double-strand break(steptr) and eggs),meiosis (Figure 5-3). In fact the exchange of reAfterformation of a synapse, the endsarefurtherprocessed by gions of homologous chromosomes,called crossing over, is nucleases, resulting in removal (stepE), andthetwo of a few bases required for proper segregationof chromosomesduring the double-stranded molecules areligatedtogether(stepB) Asa result, first the double-strand meiotic cell division. Meiosis and the consequencesof breakis repaired, but several basepairsat thesite of the breakareremoved[Adapted generating new combinations of maternal and paternal fromG Chu,1997, J Biot Chem 272:24097; M Lieber et al, 1997,Curr. geneson one chromosome by recombination are discussed OpinGenet. Devel. T:99;andD van G a n t e t a l , 2 O O 1 ,N a t u r eR e v .G e n e t 2 : 1 9 6 I further in Chapter 5. The mechanismsleading to proper segregation of chromosomes during meiosis are discussedin Chapter 20. Here we will focus on the molecular mechanisms of DNA recombination, highlighting the exchangeof DNA strands betweentwo recombining DNA molecules.

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chromosome to another. Such translocations may generare chimeric genesthat can have drastic effects on normal cell function, such as uncontrollable cell growth, which is the hallmark of cancer.The devastatingeffectsof double-strand breaks make this the "most unkindest cut of all," to borrow a phrase from Shakespeare's Julius Caesar.

H o m o l o g o u sR e c o m b i n a t i oC n a n R e p a i rD N A Damageand GenerateGeneticDiversity

150

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cHAprER 4

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Repair of a Collapsed Replication Fork An exampleof recombinationalDNA repair is the repair of a "collapsed,'replication fork. If a break in the phosphodiesterbackboneof one DNA strand is not repaired before a replication fork passes, the replicatedportions of the daughterchromosomesbecome separatedwhen the replication helicasereachesthe ,,nick,, in the parental DNA strand becausethere are no covalent bonds between the two fragments of the parental strand on either side of the nick. This processis called replication fork collapse (Figure4-41, stepn). If it is not repaired,it is generallylethal to at leastone daughtercell following cell division becauseof the loss of genetic information between the nick and the end of the chromosome.The recombination processthat repairs the resultingdouble-strandedbreak and regenerat.s r.pli."" tion fork involves multiple enzymesand other proteins, only some of which are mentionedhere.

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(Figure 4-41.,steptr ). The lagging nascentstrand (pink) created on the unnicked, homologous' parent strand is ligated to the unreplicated portion of the parent chromosome, as shown in Figure 4-41, step Z). A critical protein required for the next step is RecA in bacteria, or the homologous Rad51 in S. cereuisiae and other eukaryotes. Multiple RecA/RadS1 molecules bind to the single-stranded DNA (now considered the invading strand) and catalyze its hybridization to a perfectly or nearly perfectly complementary sequencein another, homologous, double-strandedDNA molecule, either the ligated molecule created after fork collapse (as shown in the figure) or the other homologous chromosome in diploid organisms.The other strand (dark blue) of this target double-stranded DNA (the strand not basepairing with the invading strand) is displaced as a singlestranded loop of DNA along the region of hybridization between its complement and the invading strand (refer to Figure 4-4L, step B). The RecA/RadS1-catalyzedinvasion of a duplex DNA by a single-strandedcomplement of one of the strands is key to the recombination process. Since no basepairs are lost or gained in this process,calledstrand inuasion,it doesnot requirean input of energy. Next, the hybrid region between target DNA (pink) and the invading strand (dark red) is extended in the direction away from the break by proteins that use energy from ATP hydrolysis. This process is called branch migration (Ftgure 4-41,, step 4) becausethe point at which the target DNA strand (pink) crossesfrom one complementary strand (dark blue) to its complement in the broken DNA molecule (dark red), is called a branch in the DNA structure. In this diagram, the diagonal lines represent only one phosphodiesterbond. Molecular modeling and other studiesshow that the first baseon either side of the branch is base-pairedto a complementary nucleotide. As this branch miSrates to the Ieft, the number of base pairs remains constant; one new base pair formed with the (red) invading strand is matched by the loss of one base pair with the parental (dark blue)

strand. When the region of hybridization extends beyond the 5' 3', end of the broken strand (light blue), this broken parental DNA strand becomes increasingly single-stranded, as its repairof a collapsed A FIGURE 4-41 Recombinational complement, the invading (dark red) strand' base-pairsinfork. Parental strands arelightanddarkblueTheleading replication steadwith the target (pink) DNA strand. This single-stranded pink. red, lagging daughter strand is dark and the daughter strand (light blue) parcntal strand then base-pairswith the complephosphodiester mentary region of the other parental strand (dark blue) that represent a single linesin stepB andbeyond Diagonal colorSmallblack bondfromtheDNAstrandof theconesponding has likewise become single-strandedas the branch migrates of the phosphodiester cleavage arrowsfollowingstep@ represent to the left (Figure 4-41., step 4). The resulting structure is See in the Holliday structure. of DNAstrands bondsat the crossover called a Hotliday strwcture, after the geneticist who first prornalVm bb/ruva. htmI f or an animationof sdscedu/jou http://www. posed it as an intermediatein Seneticrecombination. Again, RuvAandRuvB. See catalyzed by E coliproteins branchmigration the diagonal lines in the diagram following step 4 represent genetics.wisc.edu/Holliday/holliday3D htmlfor an http://engels single phosphodiesterbonds (not a stretch of DNA), and all Seethetextfor a of theHolliday structure anditsresolution animation basesin the Holliday structure are base-pairedto compleLehninger fromD L Nelson andM M Cox,2005, discussion. [Adapted mentary basesin the parental strands. Cleavageof the phos4thed, W H Freeman andCompany.l of Biochemistry Principles phodiesterbonds that crossover from one parental strand to ' the other and ligation of the 5' and 3 ends base-pairedto the The first step in the repair of the double-strand break is same parental strands (stepsE and 6) result in the generaexonucleolytic digestionof the strand (light blue) that has its (dark tion of a structure similar to a replication fork. Rebinding of red) 5' end at the break, leaving a portion of the other replication fork proteins results in extension of the leading (the single-stranded one with the 3' end at the break) strand D N A R E P A I RA N D R E C O M B I N A T I O N

151

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A FIGURE 4-42 Double-strand DNAbreakrepairby homologousrecombination. Forsimplicity, eachDNAdoublehelix is represented by two parallel lineswith the polarities of the strands indicated by arrowheads at their3, endsTheuppermolecule hasa double-strand breakNotethat in the diagram of the upperDNA strand past the point of the original strand break and reinitiation of lagging-strandsynthesis(step Z), thus regenerating a replication fork. The overall processallows the undamagedupper strand in the lower molecule following step E (pink/light blue) to serveas template for extensionof the leading (dark red) strand in step Z. Double-Stranded DNA Break Repair by Homologous Recombination A similar mechanism called bomolopous 152

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molecule the strandwith its3' endat the rightison thetop,whilein thediagram of the lowerDNAmolecule thisstrandisdrawnon the bottom Seethetextfordiscussion. L Orr-WeaverandJ W [AfterT. Szostak, 1985,MicrobiolRev. 49:33.1 recombination can repafua double-strand break in a chromosome and can also exchangelarge segmentsof two double-strandedDNA molecules (Figure 4-42). Homologous recombination is also dependenton strand invasion catalyzed principally by RecA in bacteriaand Rad51 in eukaryotes(steps Il and tr). The 3' end of the invading DNA strand is then extended by a DNA polymerase,displacingthe parental strand as an enlargingsingle-stranded loop of DNA (dark blue, step E). Vhen DNA synthesis extends sufficiently far, the displaced

B A s t cM o L E c u L AGRE N E TM t cE c H A N t s M s

parentalstrand (dark blue)that is complementaryto the 3' single-strandedregion generatedat the other broken end of DNA (the pink single-stranded region on the left following step n), base-pair(step B). This 3' end the complementarysequences (pink) is then extendedby a DNA polymerase,using the displaced single-strandedloop of parental DNA (dark blue) as template(step4). Next, the two 3' ends generatedby DNA synthesisare ligated(stepE)to the 5'ends generatedin step I by 5'-exonucleasedigestion of the broken ends. This generatestwo Holliday structuresin the paired molecules(stepE).Branch migration of theseHolliday structurescan occur in either direction (not diagrammed).Finally, cleavageof the strands at the positions shown by the arrows, and ligation of the alternative 5' and 3' ends at each cleavedHolliday structure generatestwo recombinant chromosomesthat each contain the DNA of one parental DNA molecule (pink and red strands) on one side of the break point and the DNA of the other parental DNA molecule (light and dark blue) on the other sideof the break point (step6). Eachchromosomecontains a third region, located in the immediate vicinity of the initial break point, that forms a heterodwplex;here one strand from one parent is base-pairedto the complementary strand of the other parent (pink or red base-pairedto dark or light blue). Base-pair mismatches between the two parental strandsare usually repairedby repair mechanismsdiscussed above to generatea complementary basepair. In the process, sequencedifferencesbetween the two parents are lost, a processreferredto as geneconuersion. Figure 4-43 diagrams how cleavageof one or the other pair of strandsat the four-way strand junction in the Holliday structure generatesparental or recombinant molecules.This process,calledresolution of the Holliday structure,separates DNA molecules initially joined by RecA/RadS1-catalyzed strand invasion. Each Holliday structure in the intermediate following step El of Figure 4-42 can be cleavedand religated in the two possibleways shown by the two setsof small black arrows. Consequently,there are four possibleproducts of the

recombination process.Two of theseregeneratethe parental chromosomes(with the exceptionof the heteroduplexregion at the break point that is repaired into the sequenceof one parent or the other (geneconversion),and t'wo generaterecombinant chromosomesas shown inFigure 4-42. Meiotic Recombination Meiosis is the specializedform of cell division in eukaryotesthat generateshaploid germ cells (e.g.,sperm and eggs)from a diploid cell (Figure 20-38). At least one recombination occurs between the paternal and maternal homologous chromosomesbefore the first meiotic cell division. Recombination is initiated by an enzyme that makes a double-strandedbreak in the DNA of one chromosome at any one of a very large number of sites.The process diagrammed in Figure 4-42 is then followed. The entire process from cleavageof the DNA of one chromosome through resolution of the Holliday structuresis repeateduntil at least one recombination, also called a crossouer'occurs between one pair of each of the homologous chromosomes. As mentioned earlier,the resulting link betweenhomologous chromosomesis required for their proper segregationduring the first meiotic cell division (seeChapter 20). As a consequence,every germ cell contains multiple recombinant chromosomes made of large segmentsof either the maternal or the oaternal chromosome.

DNA Repair and Recombination Changesin the DNA sequenceresult from copying errs and the effects of various physical and chemical agents. r Many copying errors that occur during DNA replication are corrected by the proofreading function of DNA polymerasesthat can recognize incorrect (mispaired) basesat the 3' end of the growing strand and then remove them by activity (seeFigure4-34). an inherent3'-+5' exonuclease

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153

r Eukaryotic cells have three excision-repair systemsfor correcting mispaired basesand for removing W-induced thymine-thymine dimers or large chemical adducts from DNA. Base excision repair, mismatch repair, and nucleotide excision repair operate with high accuracy and generally do not introduce errors. r Repair of double-strand breaks by the nonhomologous end-joining pathway can link segmentsof DNA from different chromosomes, possibly forming an oncogenic translocation. The repair mechanismalso producesa small deletion, even when segmentsfrom the same chromosome are joined. r Inherited defectsin the nucleotideexcision-repairpathway, as in individuals with xeroderma pigmentosum, predisposethem to skin cancer.Inherited colon cancer frequently is associatedwith mutant forms of proteins essentialfor the mismatch repair pathway. Defects in repair by homologous recombination are associatedwith inheritanceof one murant alleleof the BRCA-1 or BRCA2 gene and result in predispositionto breast and uterine cancer. r Error-free repair of double-strand breaks in DNA is accomplished by homologous recombination using the undamaged sister chromatid as a template. This processcan lead to recombination of parental chromosomesand is exploited by eukaryotes to generate genetic diversity by recombination of paternal and maternal chromosomes in developinggerm cells.

of the Cellutar Wl Viruses:Parasites GeneticSystem Virusesare obligate,intracellularparasites.They cannot reproduce by themselvesand must commandeera host cell's machineryto synthesizeviral proteins and in some casesto replicate the viral genome. RNA viruses, which usually replicate in the host-cell cytoplasm, have an RNA genome, and DNA viruses,which commonly replicate in the hostcell nucleus, have a DNA genome (seeFigure 4-1). Viral genomesmay be single-or double-stranded,dependingon the specific type of virus. The entire infectious virus particle, called a virion, consistsof the nucleicacid and an outer shell of protein that both protects the viral nucleic acid and functions in the processof host-cell infection. The simplest viruses contain only enough RNA or DNA to encode four proteins; the most complex can encode=200 proteins. In addition to their obvious importanceas causesof disease,viruses are extremely useful as researchtools in the study of basic biological processes,such as thosediscussedin this chaoter.

Most Viral Host RangesAre Narrow The surfaceof a virion contains many copies of one type of protein that binds specificallyto multiple copies of a receptor protein on a host cell. This interaction determines the

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host range-the group of cell types that a virus can infectand beginsthe infection process.Most viruses have a rather limited host range. A virus that infects only bacteria is called a bacteriophage, or simply a phage. Viruses that infect animal or plant cells are referred to generally as animal uiruses or plant uiruses.A few viruses can grow in both plants or animals and the insects that feed on rhem. The highly mobile insects serve as vectors for transferring such viruses between susceptibleplant or animal hosts. Wide host ranges are also characteristicof some strictly animal viruses,such as vesicularstomatitis virus, which grows in insect vectors and in many different types of mammals. Most animal viruses,however, do not cross phyla, and some (e.g.,poliovirus) infect only closely related speciessuch as primates. The host-cell range of some animal viruses is further restricted to a limited number of cell types because only these cells have appropriare surface receptors to which the virions can attach.

V i r a l C a p s i d sA r e R e g u l a rA r r a y so f O n e o r a Few Typesof Protein The nucleic acid of a virion is enclosedwithin a protein coat, or capsid, composed of multiple copies of one prorein or a few different proteins, each of which is encoded by a single viral gene.Becauseof this structure, a virus is able to encode all the information for making a relatively large capsid in a small number of genes.This efficient use of geneticinformation is important, since only a limited amount of DNA or RNA, and therefore a limited number of genes,can fit into a virion capsid. A capsid plus the enclosednucleic acid is called a nucleocapsid. Nature has found two basic ways of arranging the multiple capsid protein subunits and the viral genome into a nucleocapsid.In some viruses,multiple copies of a single coat protein form a helical structure that enclosesand protects the viral RNA or DNA, which runs in a helical groove within the protein tube. Viruses with such a helical nucleocapsid, such as tobacco mosaic virus, have a rodlike shape (Figure 4-44a). The other major structural type is based on the icosahedron, a solid, approximately spherical object built of 20 identical faces,each of which is an equilateral triangle (Figure 4-44b). During infection, some icosahedral viruses interact with host cell-surfacereceptors via clefts in between the capsid subunits; others interact via long fiberlike proteins extending from the nucleocapsid. In many DNA bacteriophages,the viral DNA is located within an icosahedral "head" that is attached to a rodlike "tail." During infection, viral proteins at the tip of the tail bind to host-cellreceptors,and then the viral DNA passesdown the tail into the cytoplasm of the host cell (Figure 4-44c). In someviruses,the symmetricallyarrangednucleocapsid is coveredby an external membrane,or envelope,which consistsmainly of a phospholipid bilayer but also conrains one or two types of virus-encodedglycoproteins (Figure 4-44d).

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The phospholipidsin the viral envelopeare similar to thosein the plasmamembraneof an infectedhost cell.The virai envelope is, in fact, derivedby budding from that membrane,but contains mainly viral glycoproteins,as we discussshortly.

V i r u s e sC a n B e C l o n e da n d C o u n t e d i n P l a q u eA s s a y s The number of infectiousviral particlesin a samplecan be quantifiedby a plaque assay.This assayis performedby culturing a dilute sample of viral particleson a plate covered with host cells and then counting the number of local Iesions,called plaques,that develop (Figure 4-45). A plaque developson the plate wherevera singlevirion initially infects

a singlecell. The virus replicatesin this initial host cell and then lyses(ruptures)the cell, releasingmany progenyvirions that infect the neighboring cells on the plate. After a few such cyclesof infection, enough cells are lysed to produce a visibleclear area,the plaque,in the layer of remaininguninfectedcells. Sinceall the progeny virions in a plaque are derived from a single parental virus, they constitute a virus clone. This type of plaque assayis in standardusefor bacterialand animal viruses.Plant virusescan be assayedsimilarly by counting local lesions on plant leavesinoculated with viruses. Analysis of viral mutants' which are commonly isolated by plaque assays,has contributedextensivelyto current understandingof molecularcellularprocesses.

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3. Replication-Yiral mRNAs are produced with the aid of the host-cell transcription machinery (DNA viruses)or by viral enzymes(RNA viruses).For both types of viruses, viral mRNAs are translated by the host-cell translation machinery. Production of multiple copies of the viral genome is carried out either by viral proteins alone or with the help of host-cell proteins. 4. Assembly-Yiral proteins and replicated genomesassociate to form progeny virions. 5. Release-Infected cell either ruptures suddenly (lysis), releasingall the newly formed virions at once, or disintegratesgraduallS with slow releaseof virions. Both cases lead to the death of the infected cell.

Eachplaque representscell lysis initiatedby one viral particle(agar restrictsmovement so that virus can infectonly contiguouscells)

Figure 4-46 rllustratesthe lytic cycle forT4 bacteriophage,a nonenveloped DNA virus that infects E. coli. Viral capsid proteins generally are made in large amounts becausemany

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A EXPERIMENTAL FTGURE 4-45 The plaqueassaydetermines the numberof infectiousparticlesin a viral suspension. (a)Each lesion, or plaque, whichdevelops wherea singlevirioninitially infected a singlecell,constitutes a pureviralclone.(b)plaques on a lawnof Pseudomonasfluorescens bacteriamadeby bacteriophage polytechnique of Dr Pierre Rossi, $S1 [Part(b)Courtesy Ecole F6d€rale de (LBE-EPFL) Lausanne l Replication of viralDNA Expression of virallateproteins

LyticViral Growth CyclesLeadto the Death of Host Cells Although details vary among different types of viruses,those that exhibit a lytic cycle of growth proceed through the following general stages: l, Adsorption-Virion interacts with a host cell by binding of multiple copies of capsid protein to specificreceptors on the cell surface. 2. Penetration-Yiral genome crossesthe plasma membrane. For some viruses,viral proteins packagedinside the capsid also enter the host cell.

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A FIGURE 4-46 tytic replicationcycleof a nonenveloped, bacterialvirus.E.colibacteriophage T4 hasa double-stranded DNA genomeandlacksa membrane envelope Afterviralcoatproteins at thetip of thetailin T4 interact with specific receptor proteins on the exterior of the hostcell,theviralgenomeisinjected intothe host (stepIl). Host-cell enzymes thentranscribe viral"early"genesinto mRNAs andsubsequently translate theseintoviral"early"proteins (stepZ) Theearlyproteins replicate theviralDNAandinduce expression of viral"late"proteins (stepS) The by host-cell enzymes virallateproteins include capsid andassembly proteins andenzymes thatdegrade the host-cell DNA,supplying nucleotides for synthesis of moreviralDNA Progeny virions areassembled in thecell(stepZl) (step5) whenviralproteins andreleased lysethecell.Newly liberated viruses initiateanothercycleof infection in otherhost ceils

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FIGURE4-47 Lytic replication cycle of an enveloped, animal e dN A v i r u s . R a b i evsi r u si s a n e n v e l o p evdi r u sw i t h a s i n g l e - s t r a n d R genome The structuralcomponentsof this virusare depictedat the top After a virionadsorbsto multiplecopiesof a specifichost membraneprotein(steptr), the cellengulfsit in an endosome ( s t e pE ) A c e l l u l apr r o t e i ni n t h e e n d o s o m em e m b r a n ep u m p s H* ionsfrom the cytosolinto the endosomeinterior.The resulting s c o n f o r m a t i o n cahl a n g ei n t h e d e c r e a sien e n d o s o m apl H i n d u c e a leadingto fusionof the viralenvelopewith the viralglycoprotein, r e m b r a n ea n d r e l e a soef t h e n u c l e o c a p s i d e n d o s o m al il p i db i l a y em uses into the cytosol(stepsB and 4) ViralRNApolymerase in the cytosolto replicatethe viralRNA ribonucleoside triphosphates genome(stepE) and to synthesize viralmRNAs(step6) One of the glycoprotein, which is viralmRNAsencodesthe viraltransmembrane ( E R a) s i t i s r n s e r t e idn t o t h e m e m b r a n eo f t h e e n d o p l a s m ri ce t i c u l u m

(stepZ). Carbohydrate isadded ribosomes on ER-bound synthesized t h e i n s i d e E Rl u m e na n di sm o d i f i eads t o t h el a r g ef o l d e dd o m a i n passthroughthe glycoprotein andtheassociated the membrane fuse (stepE) Vesicles with matureglycoprotern Golgiapparatus on the viralglycoprotein depositing membrane, with the hostplasma the cell domainoutside withthe largereceptor-binding cellsurface on host-cell aretranslated (step9) Meanwhile, otherviralmRNAs andviralRNA protein, matrixprotein, intonucleocapsid ribosomes with replicated areassembled polymerase GtepIE). Theseproteins nucleocapsids GtepI[), RNA(brightred)intoprogeny viralgenomic domainof viraltransmembrane withthecytosolic whichthenassociate (stepIE) Theplasma membrane in the plasma glycoproteins t h en u c l e o c a p sf oi dr ,m i n ga " b u d "t h a t m e m b r a ni sef o l d e da r o u n d (stePIE) isreleased eventually

copiesof them are requiredfor the assemblyof eachprogeny virion. In eachinfectedcell, about 100-200 T4 progenyvirions are producedand releasedby lysis. The lytic cycle is somewhatmore complicatedfor DNA virusesthat infect eukaryoticcells.In most suchviruses,the DNA genome is transported (with some associated proteins)into the cell nucleus.Once inside the nucleus,the viral DNA is transcribedinto RNA by the host'stranscription machinery.Processingof the viral RNA primary transcript by host-cell enzymesyields viral mRNA, which is transportedto the cytoplasmand translatedinto viral pro-

teins by host-cellribosomes,tRNA, and translationfactors. The viral proteins are then transported back into the nucleus,where some of them either replicate the viral DNA directly or direct cellular proteinsto replicatethe viral DNA' as in the case of SV40 discussedearlier.Assembly of the capsid proteins with the newly replicated viral DNA occurs in the nucleus,yieldingthousandsto hundredsof thousands of progenyvirions. Most plant and animal viruseswith an RNA genome do not require nuclear functions for lytic replication. In some of these viruses, a virus-encoded enzyme that enters the host

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d u r i n g p e n e t r a t i o n t r a n s c r i b e st h e g e n o m i c R N A i n t o mRNAs in the cell cytoplasm. The mRNA is directly translated into viral proteins by the host-cell translation machinery. One or more of theseproteins then produces additional copies of the viral RNA genome. Finally, progeny genomes are assembledwith newly synthesizedcapsid proteins into p r o g e n yv i r i o n si n t h e c y t o p l a s m . After the synthesisof hundreds to hundreds of thousandsof new virions has beencompleted,dependingon the type of virus and host cell, most infecredbacterialcellsand some infectedplant and animal cellsare lysed,releasingall the virions at once. In many plant and animal viral infections, however, no discretelytic event occurs; rather, the dead host cell releasesthe virions as it gradually disintegrates. As noted previously,envelopedanimal virusesare surrounded by an outer phospholipid layer derived from the plasma membrane of host cells and containing abundant viral glycoproteins. The processesof adsorption and release of envelopedviruses differ substantially from these p r o c e s s e sf o r n o n e n v e l o p e dv i r u s e s . T o i l l u s t r a t e l y t i c replication of enveloped viruses, we consider the rabies v i r u s , w h o s e n u c l e o c a p s i dc o n s i s t so f a s i n g l e - s t r a n d e d RNA genome surrounded by multiple copies of nucleocapsid protein. Like most other lytic RNA viruses,rabies virions are replicated in the cytoplasm and do not require host-cell nuclear enzymes.As shown in Figure 4-47, a rabies virion is adsorbedto a host cell by binding to a specific cell-surfacereceptor moleculeand then entersthe cell by endocytosis.Progeny virions are releasedfrom a host cell by budding from the host-cellplasma membrane.Budding virions are clearly visible in electron micrographs of infected cells, as illustrated in Figure 4-48. Many tens of thousands of progeny virions bud from an infected host cell before it dies.

V i r a l D N A l s I n t e g r a t e di n t o t h e H o s t - C e l l G e n o m ei n S o m eN o n l y t i cV i r a l G r o w t h C y c l e s Some bacterial viruses, called temperate phages,can establish a nonlytic associationwith their host cellsthat doesnot kill the cell. For example,when bacteriophageinfectsE. coli, the viral DNA may be integrated into the host-cell chromosome rather than being replicated. The integrated viral DNA, called a prophage, is replicated as part of the cell's DNA from one host-cell generation to the next. This phenomenon is referred to as lysogeny. Under certain conditions, the prophage DNA is activated,leading to its excision from the host-cell chromosome and entrance into the lytic cycle, with subsequentproduction and releaseof progeny vlrtons. The genomesof a number of animal viruses also can rntegrateinto the host-cell genome. One of the most important are the retroviruses,which are envelopedviruses with a genomeconsistingof two identical srrandsof RNA. These viruses are known as retrouiruses because their RNA genome acts as a template for formation of a DNA

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A EXPERIMENTAL FIGURE 4-48 Progenyvirionsare released by budding.Progeny virions of enveloped viruses arereleased by buddingfrominfected cellsIn thistransmission electron micrograph of a cellinfected with measles virus,virionbudsareclearly visible protruding fromthe cellsurfaceMeasles virusisan enveloped RNA viruswith a helical nucleocapsid, likerabies virus,andreplicates as illustrated in Figure4-47 [From A Levine. 1991 Scientific , Viruses, p 221 American tibrary,

molecule-the opposite flow of geneticinformation compared with the more common transcription of DNA into RNA. In the retroviral life cycle (Figure 4-49), a viral enzyme called reversetranscriptaseinitially copies the viral RNA genome into single-strandedDNA complementary to the virion RNA; the same enzyme then catalyzessynthesisof a complementaryDNA strand. (This complex reaction is detailed in Chapter 6 when we consider closely r e l a t e d i n t r a c e l l u l a r p a r a s i t e sc a l l e d r e t r o t r a n s p o s o n s . ) The resulting double-strandedDNA is integrated into the chromosomal DNA of the infected cell. Finally, the integrated DNA, called a provirus, is transcribed by the cell's own machinery into RNA, which either is translated into viral proteins or is packagedwithin virion coat proteins to form progeny virions that are releasedby budding from the host-cell membrane. Becausemost retrovirusesdo not kill their host cells, infected cells can replicate,producing daughter cells with integrated proviral DNA. These daughter cells continue to rranscribe the proviral DNA and bud progeny virions. Some retrovirusescontain cancer-causing genes (oncogenes),and cellsinfectedby suchretrovirusesare oncogenically transformedinto tumor cells.Studiesof oncogenicretroviruses (mostly virusesof birds and mice) have revealeda great deal about the processes that leadto transformationof a normal cell into a cancercell (Chapter25).

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Among the known human retrovirusesare human Tc e l l l y m p h o t r o p h i c v i r u s ( H T L V ) , w h i c h c a u s e sa form of leukemia, and human immunodeficiency virus ( H I V ) , w h i c h c a u s e sa c q u i r e d i m m u n e d e f i c i e n c y s y n drome (AIDS). Both of these viruses can infect only specific cell types, primarily certain cells of the immune system and, in the caseof HIV, some central nervous system neurons and glial cells. Only these cells have cell-surface receptors that interact with viral envelope proteins, accounting for the host-cell specificity of these viruses.Unlike most other retroviruses,HIV eventually kills its host cells. The eventual death of large numbers of immunesystem cells results in the defectiveimmune response characteristicof AIDS. Some DNA viruses also can integrate into a host-cell chromosome. One example is the human papillomaviruses (HPVs), which most commonly cause warts and other benign skin lesions. The genomes of certain HPV serotypes, however,occasionallyintegrate into the chromosomal DNA of infected cervical epithelial cells, initiating developmentof cervical cancer. Routine Pap smears can detect cells in the early stagesof the transformation processinitiated by HPV integration, permitting effectivetreatment. I

sitesin the hostintooneof manypossible andintegrated nucleus chromosome onlyonehost-cell DNA.Forsimplicity, cellchromosomal istranscribed viralDNA(provirus) isdepictedStepZl: Theintegrated (darkred)and generating mRNAs RNApolymerase, bythe host-cell (brightred).Thehost-cell machinery genomicRNAmolecules andnucleocapsid intoglycoproteins theviralmRNAs translates by andarereleased virions thenassemble proteins. StepEt: Progeny 4-47. in Figure buddingasillustrated

Viruses: Parasitesof the Cellular Genetic System r Viruses are small parasitesthat can replicate only in host cells. Viral genomes may be either DNA (DNA viruses)or RNA (RNA viruses)and either single-or doublestranded. r The capsid, which surrounds the viral genome, is composed of multiple copiesof one or a small number of virusencodedproteins. Somevirusesalso have an outer envelope' which is similar to the plasma membrane but contains viral transmembraneproteins. r Most animal and plant DNA viruses require host-cell nuclear enzymesto carry out transcription of the viral genome into mRNA and production of progeny genomes' In contrast, most RNA viruses encode enzymesthat can transcribe the RNA genomeinto viral mRNA and produce new copies of the RNA genome. r Host-cell ribosomes, tRNAs, and translation factors are used in the synthesisof all viral proteins in infected cells.

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r Lytic viral infection entails adsorption, penetration, synthesisof viral proteins and progeny genomes(replication), assemblyof progeny virions, and releaseof hundreds to thousandsof virions, leadingto death of the host cell (seeFigure 4-46).Releaseofenveloped vrrusesoccurs by budding through the host-cell plasma membrane (see Figure 4-471. r Nonlytic infection occurs when the viral genome is integrated into the host-cell DNA and generally does not lead to cell death. r Retroviruses are enveloped animal viruses containing a single-stranded RNA genome. After a host cell is penetrated, reversetranscriptase,a viral enzymecarried in the virion, converts the viral RNA genome into doublestranded DNA, which integratesinto chromosomal DNA (seeFigure4-49). r Unlike infection by other retroviruses,HIV infection eventually kills host cells, causing the defects in the immune responsecharacteristicof AIDS. r Tumor viruses, which contain oncogenes,may have an RNA genome (e.g.,human T:cell lymphotrophic virus) or a DNA genome (e.g., human papillomaviruses).In the case of theseviruses,integration of the viral genomeinto a hostcell chromosome can causetransformation of the cell into a tumor cell.

In this chapter we first reviewed the basic structure of DNA and RNA and then describedfundamental aspectsof the transcription of DNA by RNA polym.rur.t. RNA polymerases are discussed in greater detail in Chapter 7, along with additional factors required for transcriotion i n i t i a t i o n i n e u k a r y o t i cc e l l sa n d i n t e r a c t i o n sw i t h . e g u l a tory transcription factors that control transcription initiation in both bacterial and eukaryotic cells. Next, we discussedthe geneticcode and the participation of IRNA and the protein-synthesizingmachine, the ribosome, in decoding the information in mRNA to allow accurateassembly of protein chains. Mechanisms that regulate protein synthesis are consideredfurther in Chapter 8. Then, we consideredthe molecular detailsunderlying the accuratereplication of DNA required for cell division. Chapter 20 coversthe mechanismsthat regulatewhen a cell replicates its DNA and that coordinate DNA replication with the complex process of mitosis that distributes the daughter DNA molecules equally to each daughter cell. The next section addressedmechanisms for repairing damage to DNA, including recombination mechanismsthat also lead to the generationof geneticdiversity among individuals of a species.This genetic recombination contributes to the diversity of traits subjectedro natural selectionduring the evolution of contemporary species.In Chapter 20, we discuss the mechanisms that segregatechromosomes into haploid germ cells, a processthat requires recombination 160

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between maternal and paternal chromosomes.Finally, we discussed viruses, parasites of the cellular molecular genetic system and important model systemsand useful tools for studying multiple aspects of molecular cell biology. The basicmolecular geneticprocessesdiscussedin this chapter form the foundation of contemporary molecular cell biology. Our current understandingof theseprocesses is grounded in a wealth of experimentalresults and is not likely to change.However, the depth of our understanding will continue to increaseas additional details of the structures and interactionsof the macromolecularmachinesinvolved are uncovered. The determination in recent years of the three-dimensionalstructures of RNA polymerases, ribosomal subunits, and DNA replication proteins has allowed researchersto design ever more penerraung experimental approachesfor revealinghow thesemacromoleculesoperate at the molecular level. The detailed level of understanding currently being developed may allow the design of new and more effective drugs for treating illnessesof humans, crops, and livestock.For example,the recent high-resolutionstructuresof ribosomesare providing insights into the mechanism by which antibiotics inhibit bacterial protein synthesiswithout affecting the function of mammalian ribosomes. This new knowledge may allow the design of even more effective antibiotics. Similarln detailed understanding of the mechanisms regulating transcription of specifichuman genesmay lead to therapeutic strategies that can reduce or prevent inappropriate immune responsesthat lead to multiple sclerosisand arthritis, the inappropriate cell division that is the hallmark of cancer, and other pathological processes. Much of current biological researchis focusedon discovering how molecular interactions endow cells with decision-making capacity and their special properties. For this reason severalof the following chapters describecurrent knowledge about how such interactions regulate transcription and protein synthesisin multicellular organisms and how such regulation endows cells with the capacity to become specializedand grow into complicated organs. Other chapters deal with how protein-protein interactions underlie the construction of specialized organellesin cells, and how they determine cell shapeand movement. The rapid advancesin molecular cell biology in recent years hold promise that in the not too distant future we will understand how the regulation of specialized cell function, shape,and mobility coupled wirh regulared c e l l r e p l i c a t i o n a n d c e l l d e a t h ( a p o p t o s i s )l e a d t o t h e growth of complex organisms like flowering plants and human beings.

KeyTerms anticodon127 codons127 complementary L14

B A S tM c o L E c u L AGRE N E TM t cE c H A N t s M s

crossingover 150 deamination 146 depurination 147

DNA end-joining 149

polyribosomes138

DNA polymerases141

primary transcript 121

double helix 114 envelope(viral) 154

primer 141

excision-repair systems147

reading frame 1.28

exons 123 geneconversion153

recombination112

genetic code 127

retroviruses152

Holliday structure154

reversetranscriptase152

homologousrecombination repair L50

ribosomal RNA (rRNA) 112

introns 123

ribosomes127

lagging strand 141

RNA polymerase120

leading strand 141

thymine-thymine dimers 148

messengerRNA (mRNA) 112

promoter 1-21-

replicationfork 141

mutation 138

transcription1 12 transferRNA (IRNA) 112

Okazaki fragments 141 phosphodiesterbond 1 14

translation 112 'Watson-Crick basepairs 114

Review the Concepts l. \fhat are'Watson-Crickbasepairs? lWhy are they important? 2, TAIA box-binding protein binds to the minor groove of DNA, resulting in the bending of the DNA helix (seeFigure 4-5). Vhat property of DNA allows the TAIA box-binding protein to recognizethe DNA helix? 3. Preparing plasmid (double-stranded,circular) DNA for sequencinginvolves annealing a complementary,short, single-strandedoligonucleotide DNA primer to one strand of the plasmid template. This is routinely accomplishedby heating the plasmid DNA and primer to 90 "C and then 'C. \ilhy does slowly bringing the temperature down to 25 this protocol work? 4. \fhat difference between RNA and DNA helps to explain the greater stability of DNA? What implications does this have for the function of DNA? 5. What are the major differencesin the synthesisand structure of prokaryotic and eukaryotic mRNAs? 6, 'While investigatingthe function of a specificgrowth factor receptor gene from humans, researchersfound that two types of proteins are synthesizedfrom this gene. A larger protein containing a membrane-spanningdomain functions to recognizegrowth factors at the cell surface,stimulating a specific downstream signaling pathway. In contrast, a related, smaller protein is secretedfrom the cell and functions to bind availablegrowth factor circulating in the blood, thus inhibiting the downstream signaling pathway. Speculateon how the cell synthesizesthesedisparateproteins. 7. The transcription of many bacterial genesrelieson functional groups called operons,such as the tryptophan operon

'$7hat advantagesare (Figure 4-1.3a).'What is an operon? there to having genesarrangedin an operon' compared with the arrangementin eukaryotes? 8. Contrast how selectionof the translational start site occurs on bacterial,eukaryotic, and poliovirus mRNAs. 9. \fhat is the evidence that the 23S rRNA in the large rRNA subunit has a peptidyltransferaseactivity? 10. How would a mutation in the poly(A)-binding protein I gene affect translation? How would an electron micrograph of polyribosomesfrom such a mutant differ from the normal pattern? L1. What characteristic of DNA results in the requirement that some DNA synthesis is discontinuous? How are Okazaki fragments and DNA ligase utilized by the cell? L2. Eukaryotes have repair systems that prevent mutations due to copying errors and exposure to mutagens. 'What are the three excision-repair systemsfound in eukaryotes, and which one is responsible for correcting thymine-thymine dimers that form as a result of UV light damageto DNA? 13. DNA-repair systemsare responsiblefor maintaining genomic fidelity in normal cellsdespitethe high frequencywith which mutational eventsoccur.'Sfhattype of DNA mutation is generatedby (a) UV irradiation and (b) ionizing radiation? Describe the system responsiblefor repairing each of these types of mutations in mammalian cells. Postulatewhy a loss of function in one or more DNA-repair systems typifies many cancers. 14. lVhat is the name given to the processthat can repair DNA damage and generate genetic diversity? Briefly describe the similarities and differences of the two processes. 15. The genome of a retrovirus can integrate into the host-cellgenome.What geneis unique to retroviruses,and why is the protein encoded by this gene absolutely necessary for maintaining the retroviral life cycle?A number of retroviruses can infect certain human cells. List two of them, briefly describe the medical implications resulting from these infections, and describewhy only certain cells are infected.

Analyze the Data Protein synthesisin eukaryotes normally begins at the first AUG codon in the mRNA. Sometimes,however' the ribosomes do not begin protein synthesisat this first AUG but scan past it (leaky scanning),and protein synthesisbegins instead at an internal AUG. In order to understandwhat features of an mRNA affect efficiency of initiation at the first AUG, studies have been undertaken in which the synwas examined. thesisof chloramphenicolacetyltransferase protein referred a give rise to Translation of its messagecan protein, CAT smaller a slightly give rise to to as preCM or (see M. Kozak. 2005. Gene 367:1'3). The two proteins differ in that CAT lacks several amino acids found at the A N A L Y Z ET H E D A T A

T

161

N-terminus of preCAT. CAT is not derived by cleavageof preCAT but, instead, by initiation of translation of the mRNA at an internal AUG:

precAT Start

CAT Start

Stop

I

ll

vt m 7 c p p p " , ' ' , ", A U G 12

,:r [J[[*;,[[[[n

AUG

a. Resultsfrom a number of studieshave given rise to the hypothesisthat the sequence(-3)ACCAUGG(+4), in which the start codon AUG is shown in boldface,provides an optimal context for initiation of protein synthesisand ensures that ribosomesdo not scanpast this first AUG to begin initiation insteadat a downstreamAUG. In the numbering scheme used here, the A of the AUG initiation is designated(*1); bases5' of this are given negarivenumbers [so that the first b a s eo f t h i s s e q u e n cies ( - 3 ) ] , a n d b a s e s3 ' t o t h e ( + 1 ) A a r e given positive numbers [so that the last baseof this sequence is (+4)]. To test the hypothesisthat the start sire sequence (-3)ACCAUGG(+4) preventsleaky scanning,the chloramphenicol acetyltransferase mRNA sequencewas modified and the resulting effectson translation assessed. In the following figure, the sequence(red) surrounding the first AUG codon (black) of the mRNA that gives rise to the synthesisof preCAT is shown above lane 3. Modification of this messageis shown above the other gel lanes (altered nucleotides are in blue), and the completedproteins generatedfrom eachmodified messageappear as bands on the SDS-polyacrylamide gel below. The intensity of each band is an indication of the amount of that protein synthesized.Analyzethe alterations to the wild-type sequence,and describe how they affect translation. Are the positions of some nucleotidesmore important than others? Do the data shown in this figure provide support for the hypothesisthat the context in which the first AUG is presentaffectsefficiencyof translation from this site? Is ACCAUGG an optimal contexr for initiation from the first AUG?

CCCCA -3UUAAC UUCCC UUCCA (t

AUG1 I A A A A A U U U U ^-^^"-1U

PrtrLAr

G G IC +4UGGAA

preCAT+ * CAT+*

***{} s

G

G

b. What are some additional alterations to this message, other than those shown in the figure, that would further elucidate the importance of the ACCAUGG .

c H A p r E R4

|

c. A mutation causing a severe blood diseasehas been found in a single family (seeT. Matthes et a1.,2004, Blood 104:2181). The mutation, shown in red in the fig, ure below, has been mapped to the 5' untranslatedregion of the gene encoding hepcidin and has been found to alter the gene'smRNA. The shadedregions indicate the coding sequenceof the normal and mutant genes.No hepcidin is produced from the altered mRNA, and lack of hepcidin resultsin the disease.Can you provide a reasonableexplanation for the lack of synthesisof hepcidin in the family memberswho have inherited this mutation? $fhat can you deduce about the importance of the context in which the start site for initiation of protein synthesisoccurs in this c a s e?

Starthepcidin .....cCAGUGGGACAGCCAGACAGACGGcncCnricccncUG..... Norma

I

Y .....GCAAUGGGACAGCCAGACAGACGGCACGAUGGCACU........ Mutant

References Structure of Nucleic Acids Arnott, S. 2006. Historical article: DNA polymorphism and the earfy history of the double helix. TrendsBiochem. Sci.3l:349-354. Berger,J. M., and J. C. Wang. 1996. Recentdevelopmentsin DNA topoisomeraseII structureand mechanism.Curr. Opin. Struc. Biol. 6:84-90. Dickerson,R. E. 1983. The DNA helix and how it is read. Sci. Am. 249:94-1.'1.1.. Dickerson,R. E., and H. L. Ng. 2001. DNA srructurefrom A to B. Proc. Nat'l Acad. Sci.USA.98:6986-69888. Doudna, J. A., and T. R. Cech.2002.The chemicalrepertoireof natural ribozymes.N ature 418:222-228. Kornberg, A., and T. A. Baker.2005. DNA Replication.University Science,chap. 1. A good summary of the principlesof DNA structure. Lilley, D. M. 2005. Structure,folding and mechanismsof ribozymes.Curr. Opin. Struc.Biol. t5:313-323. Vicens,Q., and T. R. Cech.2005. Atomic level architectureof group I introns revealed.TrendsBiochem. Sci.3l:41-51. 'Wang, J. C. 1980. SuperhelicalDNA. TrendsBiochem. Sci. 5:279-221. Wigley,D.B. 1995. Srructureand mechanismof DNA topoisomerases.Ann. Reu.Biophys. Biomol. Struc.24:L85-208.

+

Lane12345

'162

sequence as an optimal context for synthesis of preCAT rather than CAT? How would you further examine w h e t h e rA a t t h e ( - 3 ) p o s i t i o n a n d G a t t h e ( + 4 ) p o s i t i o n are the most important nucleotidesto provide context for the AUG start?

Transcription of Protein-Coding Genes and Formation of Functional mRNA Brenner,S., F. Jacob,and M. Meselson.1,96L.An unstableintermediatecarrying information from genesto ribosomesfor protein synthesis.Natur e 190:576- 5 8'1,. Murakami, K. S., and S. A. Darst. 2003. BacterialRNA polvmerases:the whole story.Curr. Opin. Strwc.Biol. t3:31-39.

B A S t cM o L E c u L A R G E N E I cM E c H A N t s M s

Okamoto K., Y. Sugino,and M. Nomura. 1962. Synthesisand turnover of phagemessengerRNA in E. coli infectedwith bacteriophageT4 in the presenceof chloromycetin.J. Mol. Biol. 5:527-534. Steitz,T. A. 2006. Visualizingpolynucleotidepolymerase machinesat work. EMBO J.25:3458-3468. The Decoding of mRNA by tRNAs Alexander,R. !(., and P. Schimmel.2001. Domain-domain comProg. Nucl. Acid Res. munication in aminoacyl-tRNA synthetases. Mol. Biol. 69:31,7-349. Hatfield, D. L., and V. N. Gladyshev.2002.How seleniumhas altered our understanding of the genetic code.Mol. Cell Biol. 22:3565-3576. Hoagland, M. B., et al. 1958. A solubleribonucleicacid intermediatein protein synthesis./. Biol. Chem.23l:241-257. Ibba, M., and D. Soll. 2004. Aminoacyl-tRNAs: settingthe limits of the geneticcode.GenesDeu. 18:731-738. Khorana, G. H., et al. 1966. Polynucleotidesynthesisand the geneticcode. Cold Spring Harbor Symp. Quant. Biol.3l:3949. Nakanishi, K., and O. Nureki. 2005. Recentprogressof structural biology of IRNA processingand modification. MoL Cells l91.57-166. Nirenberg, M., et al. 1966.The RNA code in protein synthesis. Cold Spring Harbor Symp. Quant. Biol. 3t:ll-24. Rich. A.. and S.-H. Kim. 1978. The three-dimensionalstructure of transfer RNA. Scl.Am.240(1,1:52-62(offprint 1.377). Stepwise Synthesis of Proteins on Ribosomes Abbott, C. M., and C. G. Proud. 2004. Translationfactors:in sicknessand in health. TrendsBiocbem.Sci.2925-131. Auerbach,T., A. Bashan,and A. Yonath. 2004. Ribosomal antibiotics:structural basisfor resistance,synergismand selectivity. TrendsB iotech nol. 22:570- 576. Frank, J., et al. 2005. The role of IRNA as a molecularspring in decoding,accommodation,and peptidyl transfer.FEBSLett. 579:959-962. Ganoza,M. C., M. C. Kiel, and H. Aoki. 2002. Evolutionary conservationof reactionsin translation.Microbiol. Mol. Biol. Reu. 66:460485. Gualerzi,C. O., et al. 2001. Initiation factorsin the early events of mRNA translation in bacteria.Cold Spring Harbor Symp. Quant. Biol. 66:363-376. Hellen, C. U., and P. Sarnow.2001. Internal ribosomeentry sitesin eukaryotic mRNA molecules.Genet.Deuel. 15:1593-161'2. Kahveiian,A., G. Roy, and N. Sonenbery.200l. The mRNA closed-loopmodel: the function of PABPand PABP-interactingproteins in mRNA translation.Cold Spring Harbor Symp. Quant. Biol. 66:293-300. Kapp, L. D., and J. R. Lorsch. 2004. The molecularmechanics of eukaryotictranslation.Ann. Reu.Biochem. 73:657-704. Mitra, K., and J. Frank. 2006. Ribosomedynamics:insights from atomic structuremodeling into cryo-electronmicroscopy maps.Ann. Reu.Biophys.Biomol. Struc.35:299-3t7. Noller, H. F. 2005. RNA structure:readingthe ribosome. Science309:1508-15 14. Noller, H. F., et al. 2002. Translocationof IRNA during protein synthesis.FEBSLett. 514i1.1.-16. Polacek,N., and A. S. Mankin. 2005. The ribosomal peptidyl transferasecenter:structure,function, evolution, inhibition' Cdr. Reu.Biocbem.Mol. Biol. 4O:285-31.1. Preiss,T., and M. W. Hentze.2003. Startingthe protein synthesis machine:eukaryotic translation initiation. BioEssays 25:1201-1.211'. Richter,J. D., and N. Sonenberg.2005. Regulationof capdependenttranslation by eIF4E inhibitory proteins.Nature 433:477480.

Scheper,G. C., C. G. Proud, and M. S' van der Knaap. 2005' Defectivetranslation initiation causesvanishingof cerebralwhite matter.TrendsMol. Med. t2:1'59-1'66. Sonenberg,N. 2006. TranslationalControl in Biology and Medicine (CSH monograph).Cold SpringHarbor Press. Sonenberg,N., and T. E. Dever.2003. Eukaryotic translation initiation factors and regulators.Curr. Opin. Struc.Biol' 13:56-63' Steitz,T. A. 2005. On the structural basisof peptide-bondformation and antibiotic resistancefrom atomic structuresof the large ribosomal subunit. FEBSLett. 579:955-958' Taylor, S. S., N. M. Haste' and G. Ghosh. 2005. PKR and eIF2ct:integration of kinasedimerization,activation' and substrate docking. Cell 122:823-82 5. DNA Replication Brautigam, C. A, and T. A. Steitz. 1998' Structural and functional insights provided by crystal structures of DNA polymerases and their snbtti"t. complexes.Curr. Opin. Struc. Biol. 8:54-63. Bullock, P.A. 1997. The initiation of simian virus 40 DNA replication in vitro. Crit. Reu.Biochem.Mol. Biol' 32:503'568. DePamphilis,M. L., ed. 2006 DNA Replicationand Human Disease.Cold SpringHarbor Laboratory Press. Kornberg. A., and T. A. Baker.2005. DNA Replication' University Science. Langston,L. D., and M. O'Donnell. 2005. DNA replication: keep moving and don't mind the gap.Mol. Cells23:155-160' Mendez,J., and B. Stillman.2003. Perpetuatingthe double helix: molecularmachinesat eukaryotic DNA replication origins' 25:11'58-I1'67. BioEssays O'Donnell, M.2006. Replisomearchitectureand dynamicsin Eschericbia coli. l. B iol. Cbem. 281:106 5 3-106 56. Sclafani,R. A., R. J. Fletcher,and X. S' Chen' 2004' Two heads are better than one: regulation of DNA replication by hexameric helicases.GenesDeu. 18:2039-2045. DNA Repair and Recombination Andressoo,J. O., and J. H. Hoeijmakers'2005. Transcriptioncoupled repair and premature aging.Mutat. Res.577:179-1'94' Barnes,D. 8., and T. Lindahl. 2004. Repair and geneticconsequencesof endogenousDNA basedamagein mammalian cells'Ann' Reu.Genet.38:445476. Bell, C. E. 2005. Structureand mechanismoI Escherichiacoli RecA ATPase.Mol. Microbiol. 58:358-366. Friedberg,E. C., et al' 2006. DNA repair: from molecularmechanism to human disease.DNA Repair 5:986-996. Haber,J. E. 2000. Partnersand pathwaysrepairing a doublestrand break. Trends Genet. 16:259-264. system'Na/' Jiricny,1.2006. The multifacetedmismatch-repair Reu.Mol. Cell Biol.7:335-346. Khuu. P.A.. et al. 2005. The stacked-XDNA Holliday iunction and protein recognition.l. Mol. Recog.192234-242. Lilley, D. M., and R. M. Clegg' t993-The structureof the four--ay jonction in DNA. Ann. Reu-Biophys. Biomol' Struc' 22:299-328. Mirchandani, K. D., and A. D. D'Andrea- 2006. The Fanconi anemia/BRCAp"lh*"yt a coordinator of cross-link repail Exp' Cell Res.372:2647-2653' Mitchell, J. R., J. H. Hoeijmakers,and L. J. Niedernhofer'2003' Divide and.*qu.t, nucleotideexcisionrepair battlescancerand aging.Curr. Opin. Cell Biol.l5:232-240. Orr-Weaver,T. L., and J. I7. Szostak.1985. Fungal recombination. Microbiol. Reu. 49:33-58. Shin, D. S., et al. 2004. Structureand function of the double strand break repair machinery.DNA Repair 32863-873' '!food, R. D., M. Mitchell, and T. Lindahl. Human DNA repair Res.577:275-283. Mutat. senes. R E F E R E N C E 5o

16 3

Yoshida,K., and Y. Miki. 2004. Role of BRCA1 and BRCA2 as re-gulatorsof DNA repair, transcription, and cell cycle in responseto DNA damage.CancerSci. 95,:865-87t.

Viruses:Parasites of the CellularGeneticSystem Flint,S.J.,et al. 2000.Principles of Virology: Molecular Biology, Pathogenesis,and Control. ASM press. Hull, R. 2002. Matheuts' Plant Virology. Academic press.

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Klug, A. 1999.The tobacco mosaic virus particle: structure and assembly.Phil. Trans, R. Soc.Lond. B Biol. SZi.354:531-535. Knipe, D. M., and P. M. Howley eds. 2001. Fields Virology. Lippincott Villiams & Ifilkins. Kornberg, A., and T. A. Baker. 1992. DNA Replication,2d, ed,. W. H. Freeman and Company. Good summary of bacteriophagemolecular biology.

CHAPTER

GENETIC MOLECULAR TECHNIQUES (RNA|) mostgenesin the canbe usedto silence RNAinterference worm on the right (markedby C. elegansgenome Thetransgenic expresses dsRNAto the muscle a GFPreporterin headneurons) geneunc-\5, resultingin the potentdegradationof the unc-/5 of the worm ln contrast, mRNAand leadingto completeparalysis body the typicalsinusoidal the wild-typeworm on the leftexhibits of JohnKim] movement[Courtesy

I n previous chapters, we were introduced to the variety of I tasks that proteins perform in biological systems.Indeed' I the very field of molecular cell biology seeksto understand the molecular mechanismsof individual proteins and how groups of proteins work together to perform their biological functions. In studying a newly discoveredprotein' cell biologistsusually begin by asking three questionsabout it: what is its function, where is it located, and what is its structure?To answer thesequestions,investigatorsemploy three tools: the genethat encodesthe protein, a mutant cell line or organism that lacks the function of the protein, and a sourceof the purified protein for biochemicalstudies.In this chapter we consider various aspectsof two basic experimentalstrategiesfor obtaining all three tools (Figure5-1). The first strategy,often referred to as classicalgenetics, beginswith isolation of a mutant that appearsto be defective in some process of interest. Genetic methods then are used to identify and isolate the affected gene. The isolated genecan be manipulatedto produce large quantitiesof the protein for biochemical experiments and to design probes for studies of where and when the encoded protein is expressedin an organism.The secondstrategyfollows essentially the same stepsas the classicalapproach but in reverse order, beginning with isolation of an interesting protein or its identification based on analysis of an organism's genomic sequence.Once the corresponding gene has been isolated, the gene can be altered and then reinsertedinto an organism. In both strategies,by examining the phenoof mutations that inactivate a particutypic consequences lar gene, geneticistsare able to connect knowledge about the sequence,structure, and biochemical activity of the

encoded protein to its function in the context of a living cell or multicellular organism. An important component in both strategiesfor studying a protein and its biological function is isolation of the corresponding gene. Thus we discussvarious techniques by which res.archits can isolate, sequence'and manipulate specific re-

we discusstechniquesthat abolish normal protein function in order to analyzethe role of the protein in the cell.

OUTLIN 5.1

GeneticAnalysisof Mutationsto ldentify and StudYGenes

166

5.2

DNA Cloningand Characterization

176

5,3

UsingClonedDNA Fragmentsto StudyGene ExPression

5.4

ldentifying and LocatingHuman DiseaseGenes

5.5

Inactivatingthe Functionof Specific Genesin EukarYotes

165

> FIGURE 5-1 Overviewof two strategies for relatingthe function,location,and structureof gene products,A mutant organism isthestarting pointfor the classical geneticstrategy(greenarrows)Thereverse (orange strategy arrows) usually beginswith identification of a protein-codrng sequence by analysis of genomesequence databases In bothstrategies, the actualgeneisisolated eitherfroma DNAlibrary or by specific amplification of the genesequence from genomicDNA Oncea clonedgeneis isolated, it canbe usedto produce theencoded protein in bacterial or eukaryotic expression systems Alternatively, a clonedgenecanbe rnactivated by oneof various techniques andusedto generate mutantcellsor orqanisms

Mutant organism/cell Comparisonof mutant and wild-type function G e n e t i ca n a l y s i s Screeningof DNA library

Clonedgene DNA sequencing

to ldentifyand StudyGenes As describedin Chapter 4, the information encoded in the DNA sequenceof genesspecifiesthe sequence-and therefore the structure and function-of every protein moleculein a cell. The power of geneticsas a tool foi studying cells and organismslies in the ability of researchersto selectivelyalter every copy of just one type of protein in a cell by making a change in the gene for that protein. Genetic analysesof mutants defectivein a particular processcan reveal (a) new genes required for the process to occur, (b) the order in which gene products act in the process,and (c) whether the proteins encoded by different genes interact with one another. Before seeinghow geneticstudiesof this rype can provide insights into the mechanism of complicated cellular or developmental process,we first explain some basic genetic terms used throughout our discussion. The different forms, or variants, of a geneare referred to as alleles.Geneticistscommonly refer to the numerous naturally occurring genetic variants that exist in populations, particularly human populations, as alleles.The term mutation usually is reserved for instances in which an allele is known to have been newly formed, such as after treatment of an experimental organism with a muragen, an agent that causesa heritable changein the DNA sequence. Stricdyspeaking,the particular serof alielesfor all the genes carried by an individual is its genotype.However, this term also is usedin a more restrictedsenseto denoteiust the allelesof the

type usually denoresan allele that is present at a much higher frequency than any of the other possible alternatives. 166

.

cHAprER 5

|

Databasesearchto identify protein-codingsequence PCRisolationof corresponding gene

Expression in cultured cells

Genetic Analysis of Mutations

ff,t

Gene inactivation

M o L E c u L AG R E N E T tr cE c H N t e u E s

Protein Localization Biochemical studies Determ i nati o n of structu re

Geneticistsdraw an important distinction between the genotype and the phenotype of an organism. The phenotype refers to all the physical attributes or traits of an individual that are the consequenceof a given genotype. In practice, however, the term phenotype often is used to denote the physical consequencesthat result from just the alleles that are under experimental study. Readily observable phenotypic characteristicsare critical in the geneticanalysisof mutations.

R e c e s s i vaen d D o m i n a n tM u t a n t A l l e l e s GenerallyHave OppositeEffectson G e n eF u n c t i o n A fundamental genetic difference between experimental organismsis whether their cells ca:i::ya single set of chromosomes or two copies of each chromosome. The former are referred to as haploid; the latter, as diploid. Complex multicellular organisms (e.g., fruit flies, mice, humans) are diploid, whereasmany simple unicellular organismsare haploid. Someorganisms,notably the yeastSaccharomycescereuisiae, can exist in either haploid or diploid states. Many cancer cells and rhe normal cells of some organisms, both plants and animals) caffy more than two copies of each chromosome.However, our discussionof genetictechniques and analysis relates to diploid organisms, including dipioid yeasts. Although many different allelesof a gene might occur in different organisms in a population, any individual diploid organism will carry two copiesof eachgeneand thus at most can have two different alleles.An individual with two different allelesis heterozygousfor a gene, whereas an individual that carriestwo identical allelesis homozygousfor a gene.A recessivemutant allele is defined as one in which both alleles must be mutant in order for the mutant phenotype to be observed;that is, the individual must be homozygous for the mutant allele to show the mutant phenotype.In contrast, the phenotypic consequences of a dominant mutant allelecan be

;;-;-'*i

=

GENOTYPE I l-----t DIPLOID PHENOTYPE

Wild type

Dominant =

i M uta nt

mutant alleles 5-2 Effectsof dominantand recessive FIGURE on phenotypein diploidorganisms.A singlecopyof a dominant bothcopies whereas a mutantphenotype, to produce alleleissufficient observedin a heterozygousindividual carrying one mutant and one wild-type allele(Figure5-2). 'Whether a mutant allele is recessiveor dominant provides valuable information about the function of the affectedgene and the nature of the causativemutation' Recessivealleles usually result from a mutation that inactivates the affected gene,leadingto a partial or completelossof fwnctioz. Suchrecessivemutations may remove part of the gene or the entire genefrom the chromosome,disrupt expressionof the gene,or alter the structure of the encoded protein, thereby altering its function. Conversely,dominant alleles are often the consequenceof a mutation that causessome kind of gain of function. Such dominant mutations may increase the activity of the encodedprotein, confer a new function on it, or lead to its inappropriate spatial or temporal pattern of expression. Dominant mutations in certain genes,however,are associated with a lossof function. For instance,somegenesatehaploinswfficient, meaning that both allelesare required for normal function. Removing or inactivating a singleallele in such a gene leadsto a mutant phenotype.In other rare instancesa dominant mutation in one allelemay lead to a structuralchangein the protein that interferes with the function of the wild-rype protein encoded by the other allele. This type of mutation, referred to as a dominant-negatiue,producesa phenotype similar to that obtainedfrom a loss-of-functionmutation. Someallelescan exhibit both recessiveand dominant properties.In such cases,statementsabout whether is dominant or recessivemust specify the phenoallele an type. For example, the allele of the hemoglobin gene in humans designatedHb'has more than one phenotypicconsequence.Individuals who are homozygousfor this allele (Hb'/Hb') have the debilitating diseasesickle-cellanemia, but heterozygousindividuals (Hb'/Hb") do not have the disease.Therefore, Hb' is recessiuefor the trait of sicklecell disease.On the other hand, heterozygous(Hb'/Hb') individuals are more resistantto malaria than homozygous (Hb"/Hb") individuals, revealing that Hb' is dominant for the trait of malaria resistance.I

to causea mutantphenotype allelemustbe present of a recessive causea lossof function;dominant usually mutations Recessive causea gainof functionor an alteredfunction usually mutations

mutationsthan dominant mutations.

Segregationof Mutations in Breeding ExperimentsRevealsTheir Dominance or RecessivitY Geneticistsexploit the normal life cycle of an organism to test for the dominance or recessivityof alleles' To see how this is done, we need first to review the type of cell division that gives rise to gametes (sperm and egg cells in higher plants and animals). l7hereas the body (somatic)cells of -ort *.tl,i.ellular organisms divide by mitosis, the germ

their genesmay exist in different allelic forms' Figure 5-3 depicts the major eventsin mitotic and meiotic cell division' InLitosis DNA replication is always followed by cell division, yielding two diploid daughter cells' In meiosis oze round'of DNe replication is followed by two separatecell divisions, yieldingfour haploid (12) cells that contain only one chromosome of each homologous pair' The apportion-

A commonly used agent for inducing mutations (mutagenesis) in experimental organisms is ethylmethane sulfonate (EMS). Although this mutagen can alter DNA sequencesin severalways, one of its most common effectsis to chemically modify guanine basesin DNA, ultimately leading to the conversionof a G'C basepair into an A T basepair. Such an alteration in the sequenceof a gene,which involves only a singlebasepair, is known as a point mutation. A silent A N D s T U D YG E N E S TO IDENTIFY OF MUTATIONS ANALYSIS GENETIC

167

Focus Animation: Mitosisftttt Focus

Meiosis

MEIOTIC CELL DIVISION

Paternal homolog Maternal homolog

S o m a t i cc e l l( 2 n )

Premeioticcell l2nl

I DNA reptication

vI

v

Replicated cnromosomes (4nl

DNA reptication

Replicated cnromosomes

I

I Homologous chromosomes + align; synapsisand crossing over

v

Mitotic apparatus

Mitotic appara

;

MetaphaseI

o o o

I

I

v

v

/\

/ Ceil division \

++

D a u g h t e cr e l l s( 2 n )

o o o o

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FIGURE 5-3 Comparison of mitosisand meiosis.Both somatrc cellsandpremeiotic germcellshavetwo copies of each (2n),onematernal chromosome andonepaternal. In mitosis, the replicated chromosomes, eachcomposed of two sister chromatids, alignat the cellcenterin sucha waythatboth daughter cellsreceive a maternal andpaternal fromolog of each morphological typeof chromosome. Durinqthefirstmerctrc division, however, eachreplicated chromosome pairswith its nomotogous partnerat the cellcenter; thispairingoff isreferred Io assynapsis, andcrossing overbetweenhomoloqous

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chromosomes is evidentat this stage One replicatedchromosomeof eachmorphological type then goesinto eachdaughtercell The resultingcellsundergoa seconddivisionwithout interveningDNA replication, with the srsterchromatidsof eachmorphological type beingapportionedto the daughtercells.In the secondmeiotic divisionthe alignmentof chromatidsand their equalsegregation into daughtercellsis the sameas in mttoticdivision.The alignmentof pairsof homologouschromosomein metaphaseI is randomwith respectto other chromosomepairs,resultingln a mix of paternally and maternallyderivedchromosomes in eachdaughtercell.

(a) Segregationof dominant mutation Mutant

Wild-tYPe

Gametes

F i r s tf i l i a l g e n e r a t i o nF, . : all offspringhave mutant phenotype

Gametes S e c o n df i l i a l g e n e r a t i o nF, r : 3/aof offspringhave mutant phenotype Normal

(b) Segregationof recessivemutation Mutanl

(Figure 5-4). If the F1 progeny exhibit the mutant trait' then ih."r.r.rr"n, allele is dominant; if the F1 progeny exhibit the wild-type trait, then the mutant is recessive'Further crossing b"t*.Ln F1 individualswill also revealdifferent patternsof inheritanceaccording to whether the mutation is dominant or recessive.When F1 individuals that are heterozygousfor a dominant allele are crossedamong themselves,three-fourths of the resulting F2 progeny will exhibit the mutant trait' In contrast, when F1 individuals that are heterozygous for a recessiveallele are crossedamong themselves,only one-fourth of the resultingF2 progeny will exhibit the mutant tratt' As noted earlier,the yeast S. cereuisiae,an lmportant experimental organism' can exist in either a haploid or a iiotoid state.1n these unicellular eukaryotes' crossesbetween haploid cells can determinewhether a mutant allele is dominani or recessive.Haploid yeast cells' which carry one copy of each chromosome' can be of two different mating types k.town as a and ct. Haploid cells of opposite mating type can mate to produce a/ct diploids, which carry two of each chromosome. If a new mutation with an ob.*i., servablephenotypeis isolatedin a haploid strain, the mutant strain can be mated to a wild-type strain of the opposite mating type to produce a/o diploids that are heterozygousfor thl mutant ull.t.. ff these diploids exhibit the mutant trait, then the mutant allele is dominant, but if the diploids appear as wild-type, then the mutant allele is recessive'I7hen a/ct diploids aie placed under starvation conditions, the cells undergo -eiosfu, giving rise to a tetrad of four haploid spores' t*Jof type a and two of type ct. Sporulation of a heterozygo.rsdipiiid cell yields two sporescalryils the mutant allele ina t*o carrying the wild-type allele (Figure 5-5)' Under Wild type (type a)

Gametes

Mutant (type o)

F i r s tf i l i a l g e n e r a t i o nF, r . no offspringhave mutant phenotype Diploid cells: w i l l n o t e x h i b i tm u t a n t phenotypeif mutation is recessive

Gametes S e c o n df i l i a l g e n e r a t i o nF, r : 1/+of offspringhave mutant phenotype

Haploid spores in tetrad: 2 will be mutant 2 will be wild tYPe

5-4 Segregationpatternsof dominantand A FIGURE recessivemutationsin crossesbetweentrue-breedingstrains o f d i p l o i do r g a n i s m sA. l l t h e o f f s p r i nign t h e f i r s t( F r ) the F1 lf the mutantalleleisdominant, generation areheterozygous. ( a ) p a r t l f t he i n p h e n o t y p a e s , m u t a n t t h e e x h i b i t o f f s p r i nw g ill g i l le x h i b itth e ee , F 1o f f s p r i nw m u t a na t l l e l ei s r e c e s s i vt h a ,si n p a r t( b ) C r o s s i nogf t h e F 1 w i l d - t y pp eh e n o t y p e different alsoproduces amongthemselves heterozygotes m u t a na t lleles r e c e s s i v e a n d f d o m i n a n t s e g r e g a t i or ant i o so r intheF,qeneration

of allelesin yeast'Haploid 5-5 Segregation FIGURE cellsof oppositematingtype(i e , oneof matingtype Saccharomyces an a/ctdiploidlf ctandoneof matingtypea) canmateto produce theothercarries and allele wild-type a dominant carries onehaploid gene, resulting the same of the allele mutant a recessive trait Undercertaln the dominant diploidwillexpress heterozygous haploidspores' four of tetrad a diploidcellwillforma conditions, trait and recessive the express will tetrad the Twoof the sporesin tratt dominant the express two will

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appropriate conditions, yeastsporeswill germinate,produc_ rng vegetativehaploid srrains of both mating rypes.

(a)

p Incubate at 23 .C for 5 n

-+

C o n d i t i o n aM l u t a t i o n sC a n B e U s e dt o S t u d y E s s e n t i aGl e n e si n y e a s t

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00001000 [000t[00

VVVV

The proceduresusedto identify and isolate mutants, referred to as genettc screens,depend on whether the experimental organism is haploid or diploid and, if the latter, whether the mutation is recessiveor dominant. Genes that encode proteins essentialfor life are among the most interestingand important ones ro study. Since phenotypic expressionof mutationsin essentialgenesleadsto death of the individual, ingeniousgeneticscreensare neededto isolateand maintain organismswith a lethal mutation.

rI V V V

Colonies

Incubate at23'C

ff5fi:JJ:ru

Temperature-sensitive for growth; growth at 23., no growth at 36.

(b) Wild type

cdc28 mutants

t

t

tagenizedyeastcellsthat could grow normally at23 "C but that could not form a colony when placedat 36;C (Figure5_6a). Once temperature-sensitive murants were isolated,fu.th., analysisrevealedthat some indeed were defectivein cell divi-

l

o cdcZmutants

Cg

6 ,G

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170

o

c H A p r E5R |

MoLEcuLA GRE N E TrtEcc H N t e u E S

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FIGURE 5-6 Haploidyeastscarrying < EXPERIMENTAL lethalmutationsare maintainedat temperature-sensitive permissive temperatureand analyzedat nonpermissive cellfor temperature-sensttive screen temperature.(a)Genetic grow form (cdc) and yeast that in Yeasts mutants divisioncycle but not at 36 "C temperature) at 23 'C (permissive colonres thatblocks (nonpermissive maycarrya lethalmutation temperature) for blocksat (b)Assay colonies of temperature-sensitive celldivision. of wildin thecellcycle.Shownherearemicrographs stages specific after mutants different temperature-sensitive yeast two and type cells, for 6 h Wild-type temperature at the nonpermissive incubation sizes of buds, to grow,canbe seenwith alldifferent whichcontinue cellsin the of the cellcycleIn contrast, stages different reflecting in thecell stage at a specific a block exhibit lowertwo micrographs of a cycleThecdc28mutantsarrestat a pointbeforeemergence cellsThecdcZmutants, appearasunbudded newbudandtherefore justbeforeseparation of the mothercellandbud whicharrest (a)see (emerging cell),appearascellswith largebudslPart daughter L H H a r t w e l l ,1 9 6 -,l J B a c t e r i o9l 3 j 6 6 2 ; p a r t ( b ) f r o m L M H e r e f o r d a n d L H Hartwell.1914,J Mol Biol 84.4451

ComplementationTestsDetermineWhether Mutations Are in the Different Recessive S a m eG e n e In the genetic approach to studying a particular cellular process, researchersoften isolate multiple recessivemutaiion, that produce the same phenotype. A common test for

ganism heterozygousfor both mutations (i'e', carrying one a one b allele)will exhibit the mutant phenotype beIll.le "nd causeneither allele provides a functional copy of the gene' In contrast, if mutation a and b arein separategenes,then heterozygotescarrying a single copy of each mutant allele will not;;hibit the mutant phenotype becausea wild-type allele

theseyeastmutants did not simply fail to grow, as they might if they carried a mutation affecting generalcellular metabolism. Rather,at the nonpermissivetemperature,the mutants of interest grew normally for part of the cell cycle but then arrestedat a particular stageof the cell cycle,so that many cells at this stagewere seen(Figure 5-6b). Most cdc mutations in that is, when haploid cdc strainsare mated yeastare recessive; to wild-type haploids,the resulting heterozygousdiploids are nor defectivein cell division. neither temperature-sensitive

R e c e s s i vLee t h a lM u t a t i o n si n D i p l o i d sC a n B e l d e n t i f i e db y I n b r e e d i n ga n d M a i n t a i n e d in Heterozygotes In diploid organisms,phenotypesresulting from recessive mutations can be observedonly in individuals homozygous for the mutant alleles.Sincemutagenesisin a diploid organism typically changesonly one allele of a gene' yielding heterozygousmutants, geneticscreensmust include inbreeding stepsto generateprogeny that are homozygous for the mutanl alleles. The geneticist H. Muller developed a general and efficient procedure for carrying out such inbreeding experiments in the fruit fly Drosophila. Recessivelethal mutaiions in Drosophila and other diploid organisms can be maintained in heterozygousindividuals and their phenotypic consequencesanalyzedin homozygotes. The Muller approach was used to great effect by C' Niisslein-Volhard and E. \Tieschaus, who systematically lethal mutations affectingembryogenesis screenedfor recessive in Drosophila. Dead homozygousembryos carrying recessive lethal mutations identifiedby this screenwere examinedunder the microscope for specific morphological defects in the embryos.Current understandingof the molecularmechanisms underlyingdevelopmentof multicellularorganismsis based,in large part, on the detailedpicture of embryonic development rWe revealedby characterizationof theseDrosoprila mutants. will discusssome of the fundamental discoveriesbased on thesegeneticstudies\n Chapter 22.

lar characterization of the CDC genes and their encoded proteins, as describedin detail in Chapter 20, has provided a ?r"-.*ork for understanding how cell division is regulated in organismsranging from yeast to humans'

Double Mutants Are Useful in Assessing t h e O r d e ri n W h i c h P r o t e i n sF u n c t i o n Based on careful analysis of mutant phenotypes associated with a particular cellular process' researchersoften can deduce the order in which a set of genesand their protein products function. Two general types of processesare amenable to such analysis: (a) biosynthetic pathways in which a precursor material is convertedvia one or more intermediatesto a final product and (b) signaling pathways that regulate other processesand involve the flow of information rather than chemical intermediates.

A N D S T U D YG E N E S TO IDENTIFY OF MUTATIONS ANALYSIS GENETIC

171

> EXPERIMENTA FLI G U R E5 - 7 Complementation analysisdetermines whether recessivemutations are in the same or different genes. Complementation testsin yeastare performedby matinghaploida and o cellscarryingdifferentrecessrve mutations to producediploidcells In the analysis of cdc mutations,pairsof differenthaploid temperature-sensitive cdc strainswere systematically matedand the resultingdiploids testedfor growth at the permissive and nonpermrsstve temperaturesIn this hypothetical example,the cdcX and cdcy mutants c o m p l e m e net a c ho t h e ra n d t h u s h a v e mutationstn differentgenes,whereasthe cdcX and cdcZmutantshavemutationsin the s a m eg e n e

M a t e h a p l o i d so f oppositemating types and carryingdifferent recessivetemperaturesensitivecdc mutations

Mutant (type a)

Mutant (type cr)

(cdcX)

(cdcY\

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\_-/ \/

Mutant (typea)

Mutant (typeo)

\cdcx)

(cdcz)

\_-./

\_-,i

\/

\r'

cdcXlcdcY (type a/o)

\r'

cdcXlcdcZ (type a/o) P l a t ea n d i n c u b a t e at permissive temperature

T e s tr e s u l t i n gd i p l o i d s for a temperaturesensitivecdc phenotype

2C

aa

Growth

No growth

6A

PHENOTYPE: Wild type INTERPRETATION:

36'C

Growth indicatesthat mutations cdcX and cdcY are in differentgenes

Y9

M utant Absenceof growth indicatesthat mutations c d c Xa n d c d c Z a r e i n t h e s a m eg e n e -Ir-V----l...t-^_ia"'-l___LF

Respectivewild-typealleles p r o v i d en o r m a lf u n c t i o n

next. In E. coLi,the genesencodingtheseenzymeslie adja_ cent to one another in the genome, constituting the trp operon (seeFigure 4-73a). The order of action of the differ_ ent genesfor theseenzymes,hencethe order of the biochemi_ cal reactions in the pathway, initially was deducedfrom the types of intermediatecompoundsthat accumulatedin each

In Chapter 14 we discussthe classicuse of the double_ mutant.strategyto help elucidatethe secretorypathway. In this pathway proteins to be secreredfrom the cell rnou. i.otheir site of synthesison the rough endoplasmicreticulum (ER) to the Golgi complex. then to ,..r.toiy vesicles, and fi_ n a l l y t o r h ec e l l s u r f a c e . Ordering of Signaling pathways As we learn in later chapters,expressionof many eukaryoticgenesis regulated by signaling pathways that are initiateJ by extracellular 172

.

c H A p r E5R | M o L E c u L AGRE N E Tr tEcc H N t o u E S

B o t h a l l e l e sn o n f u n c t i o n a l

hormones, growth factors, or other signals. Such signaling pathways may include numerouscomponents,and doublemutant analysisofren can provide insight into the functions and interactionsof thesecompon.nrr.th. only prerequisite for obtaining useful information from this type of analysisis that the two mutations must have opposite effects on the output of the sameregulatedpathway. Most commonl5 one mutatio_nrepressesexpressionof a particular reporrer gene even when the signal is present, while another mutation resultsin reporter geneexpressionevenwhen the signalis absent(i.e.,constirutiveexpression).As illustratedin Figure5_gb, two simple regulatory mechanismsare consistentwith such

Note that this technique differs from complementation analysisjust describedin that when testingtwo recessive mu_ tations, the double mutant created is homozygolzsfor both mutations.Furthermore,dominant mutantscan be subiected t o d o u b l e - m u t a natn a l v s i s .

(a)Analysisof a biosyntheticpathway 1. A m u t a t i o ni n A a c c u m u l a t e isn t e r m e d i a t e e. A m u t a t i o ni n B a c c u m u l a t e isn t e r m e d i a t 2 OF PHENOTYPE D O U B L EM U T A N T

A d o u b l em u t a t i o ni n A a n d B a c c u m u l a t e s 1. intermediate INTERPRETATION: The reaction catalYzedby A precedes the reaction catalYzedbY B. I

: '

Eo'Et'E (b)Analysisof a signalingpathway

mutant allelewould have a mutant phenotype(Figure5-9a)' The observation of genetic suppressionin yeast strains carrying a mutant actin allele (act1-1) and a secondmutation (sic5) in another gene provided early evidence for a

A mutation in A gives repressedreporterexpresslon. A mutation in B gives constitutivereporterexpression. PHENOTYPE OF : D O U B L EM U T A N T : A d o u b l em u t a t i o ni n A a n d B g i v e s repressedreporterexPression. I , INTERPRETATION: A positively regulates repofter expression and is negativelYregulated bY B' .

(a)Suppression GenotyPe AB Phenotype Wildtype

aB Mutant

ab Ab Mutant SuPPressed

rNrERPRErAloP H E N O T Y PO EF D O U B L EM U T A N T

INr ERPRErArIoN:

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A d o u b l em u t a t i o ni n A a n d B g i v e s constitutivereporterexpression.

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B

(b) Synthetic lethality 1 Genotype

AB Phenotype Wild type

5-8 Analysisof doublemutantsoften canorderthe FIGURE in stepsin biosyntheticor signalingpathways'Whenmutations process but have genesaffectthe samecellular two different of the doublemutant phenotypes, the phenotype different distinctly genes mustfunction(a) which the two in the order canoftenreveal pathway, a thataffectthe samebiosynthetic Inthe caseof mutations t em e d t a t e l y t i l la c c u m u l attheei n t e r m e d i ai m d o u b l em u t a nw in the by the proteinthatactsearlier preceding the stepcatalyzed of a signaling analysis organism(b)Double-mutant wild-type on effects haveopposite if two mutations pathwayispossible phenotype gene In thiscase,the observed of a reporter expression aboutthe orderin which information of the doublemutantprovides regulators or negative actandwhethertheyarepositive the proteins

G e n e t i cS u p p r e s s i o an n d S y n t h e t i cL e t h a l i t y t roteins C a n R e v e a Il n t e r a c t i n go r R e d u n d a n P Two other types of genetic analysis can provide additional clues about how proteinsthat function in the samecellular processmay interactwith one anotherin the living cell. Both of thesemethods,which are applicablein many experimental organisms,involve the use of double mutants in which the phenotypic effects of one mutation are changed by the presenceof a secondmutation. Suppressor Mutations The first type of analysisis basedon geneticsuppression.To understandthis phenomenon,suppose

aB Partial defect

ab

Ab

Severe defect

Partial defect

rNrERPRErArto-

,fii\

( | 11I ) U,/

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i

(cl Synthetic lethality 2 Genotype

AB

Phenotype Wildtype

aB Wild type

ab

Ab Wild tYPe

or 5-9 Mutationsthat resultin geneticsuppression A FIGURE proteins' redundant or interacting reveal syntheticlethality proteins with two defective (a)Observation thatdoublemutants phenotype butthatsinglemutantsgive (A andB)havea wild-type thatthefunctionof eachprotein indicates a mutantphenotype thatdouble other.(b)Observation the with on interaction depends mutants single than defect phenotypic severe more a have mutants (e g , subunits of a heterodimer) thattwo proteins alsoisevidence (c)Observation thata double to functionnormally. mustinteract mUtantisnonviab|ebutthatthecorrespondingsing|emutants f unctionin thattwo proteins indicates phenotype thewild-type product an essential to produce pathways redundant

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173

direct interaction in vivo between the proteins encoded by the two genes.Later biochemical studies showed that these two proteins-Act1 and Sac6-do indeed inreract in the construction of functional actin structureswithin the cell. Synthetic Lethal Mutations Another phenomenon,called synthetic lethality, produces a phenotypic effect opposrte to that of suppression.In this case,the deleteriouseffectof one mutation is greatly exacerbated(rather than suppressed)by a secondmutation in a relatedgene.One situation in which such synthetic lethal mutations can occur is illustrated in Figure 5-9b. In this example,a heterodimericprotein is partiall5 but not completely,inactivatedby mutations in eiiher one of the nonidentical subunits.However, in double mutants carrying specificmurationsin the genesencodingboth subunits,little interaction betweensubunits occurs,iesulting in severephenotypic effects. Synthetic lethal mutations

product cannor be synthesizedand the double mutanrs will be nonviable.

(a)

G e n e sC a nB e l d e n t i f i e db y T h e i r M a p p o s i t i o n on the Chromosome The preceding discussionof genetic analysisillustrates how a geneticistcan gain insight into gene function by observing the phenotypic effectsproduced by joining together different combinations of mutant allelesin the samecell or organism. For example, combinations of different alleles of the same gene in a diploid can be used to determine whether a mutation is dominant or recessiveor whether two different recessive mutations are in the same gene.Furthermore, combinations of mutations in different genes can be used to determine the order of gene function in a pathway or to identify functional relationships between genessuch as suppressionand synthetic enhancement.Generally speaking,all these methods can be viewed as analytical tests baseJ on gene functioz. \7e will now consider a fundamentally different type of genetic analysis based on gene positioz. Studies designedto determine the position of a gene on a chromosome, often referred to as genetic mapping studies, can be usedto identify the geneaffected by a particular mutation or to determine whether two mutations are in the samegene. In many organisms generic mapping studies rely on exchangesof genetic information that occur during meiosis. As discussedin Chapter 4 and as shown in Figure 5-10a,

(b)Consider two linkedgenesA and Bwith recessive allelesa and b.

-'-lf m2

Replicated chromosomes (4nl

Crossof two mutantsto construct a doublyheterozygous strain:

and over

AlAblb

x alaBlB

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A i

A n a p h a s eI

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a

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v AbaBABab abuA"brb \___________-Y_

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Recombinanttypes

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Parental gamete

tn

Recombinantgametes

tn

parental gamete

A FIGURE5-10 Recombinationduring meiosis can be used to map the position of genes. (a)Shown is an individualthat carries two mutations,designatedm/ (yellow)andm2 (green),that are on the maternaland paternalversionsof the samechromosomelf crosslngoveroccursat an intervalbetweenml andm2 beforethe first meloticdivision,then two recombinantgametesare produced; one carriesboth m / and m2, whereasthe other carriesneither mutation The longerthe distancebetweentwo mutationson a 174

.

cHAprER s

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M o l E c u L A RG E N E T tTcE C H N t o u E S

GeneticdistancebetweenA and B can be determinedfrom frequencyof parentaland recombinantgametes: gametes Geneticdistancein cM = 100, Itotbi.nant total gametes chromatid,the more likelythey are to be separatedby recombination and the greaterthe proportionof recombinantgametesproduced (b) In a typicalmappingexperiment,a strainthat is heterozygous for two differentgenesis constructedThe frequencyof parentalor recombinantgametesproducedby thls straincan be determjned from the phenotypesof the progenyin a testcross to a homozygous recessive strain The geneticmap distancein centimorgans (cM) is givenas the percentof the gametesthat are recombinant

genetic recombination takes place before the first meiotic cell division in germ cells,when the replicatedchromosomes of each homologous pair align with each other. At this time, homologous DNA sequenceson maternally and paternally derived chromatids can exchangewith each other, a process 'We now know that the resulting known as crossing over. crossovers between homologous chromosomes provide structural links that are important for the proper segregation of pairs of homologous chromatids to opposite poles during the first meiotic cell division (for discussionseeChapter 20). Considertwo different mutations,one inheritedfrom each parent, that arc located close to one another on the same chromosome.Two different typesof gametescan be produced according to whether a crossoveroccurs betweenthe mutations during meiosis. If no crossoveroccurs between them, gametesknown as parental types,which contain either one or the other mutation, will be produced. In contrast' if a crossoveroccurs betweenthe two mutations' gametesknown as recombinant types will be produced. In this example recombinant chromosomeswould contain either both mutations, or neither of them. The sites of recombination occur more or lessat random along the length of chromosomes;thus the closertogethertwo genesare, the lesslikely that recombination will occur between them during meiosis. In other words, the lessfrequently recombination occursbetweentwo genes on the same chromosome, the more tightly they are Iinked and tbe closer together they are. Two genesthat are sufficiently close together such that there are significantly fewer recombinant gametes produced than parental gametes are consideredto be geneticallylinked. The technique of recombinational mapping was devised in 1911 by A. Sturtevantwhile he was an undergraduate working in the laboratory of T. H. Morgan at Columbia University. Originally used in studies on Drosophila, this technique is still used today to assessthe distance between two genetic loci on the same chromosome in many experlmental organisms. A typical experiment designedto determine the map distancebetweentwo geneticpositions would involve two steps. In the first step' a strain is constructed that carriesa different mutation at eachposition, or locus. In to dethe secondstep, the progeny of this strain are assessed termine the relative frequency of inheritance of parental or recombinant types.A typical way to determinethe frequency of recombination between two genesis to cross one diploid parent heterozygousat each of the genetic loci to another parent homozygousfor each gene.For such a cross,the proportion of recombinant progeny is readily determined becauserecombinant phenotypeswill differ from the parental phenotypes.By convention, one genetic map unit is defined as the distance between two positions along a chromosome that results in one recombinant individual in 100 total progeny. The distance corresponding to this 1 percent recombination frequency is called a centimorgan (cM) in honor of Sturtevant'smentor,Morgan (seeFigure 5-10b). A complete discussion of the methods of genetic mapping experiments is beyond the scope of this introductory discussion;however,two featuresof measuringdistancesby recombination mapping need particular emphasis.First, the

frequency of genetic exchange between two loci is strictly proportional to the physical distancein basepairs separating ih.- o.tly for loci that are relatively close together (sa5 less than about 10 cM). For loci that are farther apart than this' a distance measured by the frequency of genetic exchange tends to underestimatethe physical distance becauseof the possibility of two or more crossoversoccurring within an inie.ual. In the limiting casein which the number of recombinant types will equal the number of parental types, the two loci under considerationcould be far apart on the same chromosome or they could be on different chromosomes, and in such casesthe loci are consideredto be unlinked' A secondimportant concept neededfor interpretation of

recombination frequency (i.e.' a genetic distance of 1 cM) representsa physical distanceof about 2.8 kilobasesin yeast distance of about 400 kilobases in compared *lth " Drosophila and about 780 kilobasesin humans' One of the chief uses of genetic mapping studies is to

human diseasescan be identified using such methods' A second general use of mapping experiments is to determine *hethe. two different mutations are in the samegene' If two mutations are in the same gene, they will exhibit tight linkage in mapping experiments,but if they are in different g..r.{ they will usually be unlinked or exhibit weak linkage'

Genetic Analysis of Mutations to ldentify and Study Genes r Diploid organisms carry two copies (alleles)of each gene,whereashaploid organismscarry only one copy' r Recessivemutations lead to a loss of function, which is masked if a normal allele of the gene is present' For the mutant phenotype to occur' both alleles must carry the mutatl0n. r Dominant mutations lead to a mutant phenotype in the presenceof a normal allele of the gene.The phenotypesassociatedwith dominant mutations often representa gain of function but in the caseof some genesresult from a loss of function. r In meiosis,a diploid cell undergoesone DNA replication and two cell divisions, yielding four haploid cells in which maternal and paternal alleles are randomly assorted (see Figure5-3).

GENES G E N E T I CA N A L Y S I SO F M U T A T I O N ST O I D E N T I F YA N D S T U D Y

175

r Dominant and recessivemutations exhibit characteristic segregatlonpatternsin geneticcrosses(seeFigure5_4). r I n h a p l o i d y e a s t .t e m p e r a t u r e - s e n s i t i vmeu t a t i o n s a r e particularly useful for identifying and studying genesessential to survival. r The number of functionally related genesinvolved in a process can be defined by complemenration analysis (see Figure-5-7). r The order in which genesfunction in a signaling pathway can be deducedfrom the phenotype of double mutants defectivein two stepsin the affectedprocess. r Functionally significant interactions between Drorelns can be deduced from the phenotypic effects o? allelespecificsuppressormutations or syntheticlethal mutations. r Genetic mapping experimentsmake use of crossingover between homologous chromosomes during meiosis to measurethe genetic distance between two different muta_ tions on the samechromosome.

DNACloningand Characterization

f[

is simply any DNA molecule composed of sequencesde_ rived from different sources. The key to cloning a DNA fragment of interest is to link it to a vector DNA molecule that can replicate within a host cell. After a singlerecombinant DNA molecule,composedof a vector plus an insertedDNA fragmenr, is introdu.id irrto " host cell, the inserted DNA is replicated along with the vec_ tor, generating a large number of identical DNA molecules. The basic schemecan be summarizedas follows:

vectorsexist, our discussionwill mainly focus on plasmid vectors in E. coli host cells,which are commonly used. Various techniquescan then be employed to identify the sequenceo{ interest from this collection of DNA fragments,known as a DNA library. Once a specificDNA fragment is isolated,it is typically characterizedby determining the exact sequenceof nucleotidesin the molecule. We end with a discussionof the polymerasechain reaction (PCR).This powerful and versatile techniquecan be usedin many ways to generatelarge quantities of a specificsequenceand otherwisemanipulate ONR m the laboratory.The various usesof cloned DNA frasmentsare discussed in subsequent sections.

RestrictionEnzymesand DNA Ligases A l l o w I n s e r t i o no f D N A F r a g m e n t si n t o CloningVectors A major objective of DNA cloning is to obtain discrete, small regions of an organism'sDNA that constitute specific genes.In addition, only relatively small DNA moleculescan be cloned in any of the availablevectors.For thesereasons, the very long DNA molecules that compose an organism's genome must be cleavedinto fragments that can be inserted into the vector DNA. Two types of enzymes-restriction enzymes and DNA ligases-facilitate production of such recombinantDNA molecules. Cutting DNA Molecules into Small Fragments Restric_ tion enzymesare endonucleasesproduced by bacteria that typically recognizespecific 4- to 8-bp sequences,calledrestrictin sites, and then cleaveboth DNA strandsat this site. Restriction sites commonly are short palindromic sequences;that is, the restriction-sitesequenceis the sameon each DNA strand when read in the 5'-+3' direction (Figure5-11). For each restriction enzyme) bacteria also produce a modification enzyme, which protects a bacterium's own DNA from cleavageby modifying it at or near eachpotenrial cleavagesite. The modification enzymeadds a methyl group to one or two bases,usually within the restriction site. When

Vector + DNA fragment EcoRl

J

+

RecombinantDNA

J

J'

r(-

|

J

Cleavage EcoRl I

Isolation, sequencing,and manipulation of purified DNA fragment Although investigatorshave devisednumerous experimental variations, this flow diagram indicatesthe essentialsteDsin DNA cloning. In this section,we first describemethods for isolating a specificsequenceof DNA from a seaof other DNA sequences. This processoften involvescutting the genomernto fragmentsand then placing each fragment ii a vector so that the entire collection can be propagatedas recombinantmole_ cules in separatehost cells. While many different types of '176 .

c H A p r E5R I M o L E c u L AGRE N E Tr tEcc H N t e u E s

|

ti +

Replication of recombinant DNA within host cells

Stickyends

s',.),

-------r

G . TTAA

c ----r

3'

\l

5

FIGURE 5-11 Cleavage of DNAby the restrictionenzyme EcoRf.Thisrestriction enzymefromE colimakesstaqqered cutsat the specific 6-bppalindromic sequence shown,yielding fragments withsingle-stranded, "sticky,, complementary ends.Manyother restriction enzymes alsoproduce fragments with stickyends

a methyl group is presentthere, the restriction endonuclease is prevented from cutting the DNA. Together with the restriction endonuclease,the methylating enzyme forms a restriction-modification system that protects the host DNA while it destroysincomingforeign DNA (e.g.,bacteriophage DNA or DNA taken up during transformation) by cleaving it at all the restriction sitesin the DNA. Many restriction enzymesmake staggeredcuts in the two DNA strands at their recognition site, generatingfragments that have a single-stranded"tail" at both ends,sticky ends (seeFigure 5-11). The tails on the fragmentsgeneratedat a given restriction site are complementaryto those on all other fragments generated by the same restriction enzyme. At room temperature, these single-strandedregions can transiently base-pairwith thoseon other DNA fragmentsgenerated with the same restriction enzyme. A few restriction enzymes,such as Alul and SmaI, cleaveboth DNA strandsat the same point within the restriction site, generating fragmentswith "blunt" (flush)endsin which all the nucleotides at the fragment ends are base-pairedto nucleotides in the complementarystrand.

The DNA isolated from an individual organism has a specificsequence,which purely by chancewill contain a specific set of restriction sites' Thus a given restriction enzyme will cut the DNA from a particular source into a reproducible set of fragmentscalled restriction fragments.The frequency with which a restriction enzymecuts DNA, and thus the average size of the resulting restriction fragments, depends largely on the length of the recognition site. For example, a restriction enzyme that recognizesa 4-bp site will cleaveDNA an averageof once every4", or 256, basepairs, whereas an enzyme that recognizesan 8-bp sequencewill cleaveDNA an averageof onceevery4E basepairs (=65 kb)' Restriction enzymes have been purified from several hundred different speciesof bacteria, allowing DNA molecules to be cut at a large number of different sequencescorresponding to the recognitionsitesof theseenzymes(seeTable5-1)' Inserting DNA Fragments into Vectors DNA fragments with either sticky endsor blunt endscan be insertedinto vector DNA with the aid of DNA ligases.During normal DNA replication, DNA ligase catalyzestheend-to-endioining (ligation) of

ENZYME

MICRO()RGANISM SOURCE

SITERECOGNITION

BamHl

B a cillu s amy lo liq u efaciens

-G-G-A-T-C.C. -C-C-T-A-G-G.

PBODUCED ENOS

J Sticky

1 J Sau3A

aureus Staphylococcus

-G-A-T-C.C-T-A-G-

Sticky

t EcoRl

Escherichia coli

Hindlll

Haemophilus influenzae

J -G-A-A-T-T-C-C.T-T-A-A-G t J -A,A-G.C-T-T.

Sticky

Sticky

-T-T-C-G-A-4.

T Smal

Serratia mdrcescens

J -C-C-C-G-G-G-G-G-G-C-C-C-

Blunt

1

Notl

No cardia otiti di s-cauiarum

-G-C_G-G.C-C-G-C-C-G-C-C-G-G-C-G-

Sticky

t Many of these recognition sequencesare included in a common polylinker sequence(seeFigure 5-13). D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N

177

short fragments of DNA called Okazaki fragments. For purposesof DNA cloning, purified DNA ligaseis usedto covalently join the ends of a restriction fragment and vector DNA that have complementaryends (Figure5-12). The vector DNA and restriction fragment are covalently ligated rogetherthrough the standard3'-+5' phosphodiesterbonds of DNA. In addition to Iigating complementary sticky ends, the DNA ligasefrom bacteriophage T4 can ligate any two blunt DNA ends. However, blunt-end ligation is inherently inefficient and requires a higher concentration of both DNA and DNA ligasethan doesligation of sticky ends.

E. coliPlasmidVectorsAre Suitablefor Cloning lsolatedDNA Fragments Plasmidsare circular, double-strandedDNA (dsDNA) molecules that are separatefrom a cell's chromosomal DNA. These extrachromosomal DNAs, which occur naturally in bacteria and in lower eukaryotic cells (e.g.,yeast),exist in a

Genomic DNA fragments (a)

3' P-AATT oH__r-----::-.---r 5,

VectorDNA (a')

(b)

s', oH 3'r---------r-TTAA-p

+

P-C GHO-:5'

3' (c)

P-A G CT HOj-:-:r

3' 5,

I

I Complementary I endsbase-pair I

v

OHP

lltr 3'r------]-TTAA

/\ PHO 2 ATP T4 DNA ligase 2AMP+2PPi (a')

5'I-AATT

(a)

3',---------F++ii

3'

s,

FIGURE 5-12 Ligationof restrictionfragmentswith complementary stickyends.In thisexample, vectorDNAcutwith EcoRl ismixedwith a sample containing restriction fragments produced by cleaving genomicDNAwith several different restriction enzymes Theshortbasesequences composing the stickyendsof eachfragment typeareshownThestickyendon the cutvectorDNA (a')base-pairs onlywith thecomplementary stickyendson theEcoR/ fragment(a)in the genomic sample. Theadjacent 3, hydroxyl and5, phosphate groups(red)on the base-paired fragments thenare joined(ligated) covalently byT4 DNAligase. 178

.

c H A p r E R5

|

MoLEcuLAR G E N E T trcE c H N t o u E s

Polylinker

Plasmid cloning vector

FIGURE 5-13 Basiccomponentsof a plasmidcloningvector that can replicatewithin an E. coli cell. plasmid vectorscontaina genesuchasamp',whichencodes selectable the enzyme andconfers resistance to ampicillin. Exogenous P-lactamase DNAcan be inserted intothe bracketed regionwithoutdisturbing theabilityof the plasmidto replicate or express theamp,gene plasmid vectors alsocontaina replication origin(ORl) sequence whereDNA replication isinitiated by host-cell enzymes. Inclusion of a synthetic polylinker containing the recognition sequences for several different restriction enzymes increases the versatility of a plasmidvectorThe vectorisdesigned sothateachsitein the polvlinker is unioueon t h ep l a s m i d .

parasitic or symbiotic relationship with their host cell. Like the host-cell chromosomal DNA, plasmid DNA is duplicated before every cell division. During cell division, copies of the plasmid DNA segregateto each daughtercell, assuring continued propagation of the plasmid through successive generationsof the host cell. The plasmidsmost commonly usedin recombinant DNA technology are those that replicate in E. col/. Investigators have engineeredtheseplasmids to optimize their use as vectors in DNA cloning. For instance, removal of unneeded portions from naturally occurring E. coli plasmids yields plasmid vectors, (=1.2-3 kb in circumferential length, that contain three regions essentialfor DNA cloning: a replication origin; a marker that permits selection,usually a drugresistancegene;and a region in which exogenousDNA fragments can be inserted (Figure 5-13). Host-cell enzymes replicate a plasmid beginning at the replication origin (ORI), a specific DNA sequenceof 50-100 basepairs. Once DNA replication is initiated at the ORI, it continues around the circular plasmid regardlessof its nucleotide sequence.Thus any DNA sequenceinsertedinto such a plasmid is replicated along with the rest of the plasmid DNA. Figure 5-14 outlines the general procedure for cloning a DNA fragment using E. coli plasmid vectors. \lhen E. coli cells are mixed with recombinant vector DNA under certain conditions, a small fraction of the cells will take up the plasmid DNA, a process known as transformation. Typically, 1, cell in about 10,000 incorporates a single plasmld DNA molecule and thus becomes transformed. After plasmid vectors are incubated with E. coli, those cells that take up the plasmid can be easily selectedfrom the much larger number of cells. For instance,if the plasmid carries a gene that confers resistance to the antibiotic ampicillin,

> EXPERIMENTAL FIGURE 5-14 DNAcloningin a plasmid vector permitsamplificationof a DNAfragment.A fragmentof an intoa plasmid vectorcontaining DNAto be clonedisfirstinserted gene(amp'),suchasthatshownin Figure 5-13 ampicillin-resistance molecule by incorporation of a plasmid Onlythefew cellstransformed cells, the mediumIntransformed willsurvive on ampicillin-containing plasmid in intodaughter cells,resulting DNAreplicates andsegregates formation of an ampicillin-resistant colonv

transformed cells can be selected by growing them in an ampicillin-containing medium. DNA fragments from a few base pairs up to =10 kb commonly are insertedinto plasmid vectors. When a recombinant plasmid with an inserted DNA fragment transforms an E. coli cell, all the antibiotic-resistantprogeny cells that arise from the initial transformed cell will contain plasmids with the sameinsertedDNA. The insertedDNA is replicated along with the rest of the plasmid DNA and segregatesto daughter cells as the colony grows. In this way, the initial fragment of DNA is replicated in the colony of cells into a large number of identical copies. Since all the cells in a colony arise from a single transformed parental cell, they constitute a clone of cells, and the initial fragment of DNA inserted into the parental plasmid is referred to as cloned DNA or a DNA clone. The versatility of an E. coli plasmid vector is increased by the addition of a polylinker, a synthetically generated sequencecontaining one copy of severaldifferent restriction sitesthat are not presentelsewherein the plasmid sequence (seeFigure 5-13). \fhen such a vector is treated with a restriction enzyme that recognizesa restriction site in the polylinker, the vector is cut only once within the polylinker. Subsequentlyany DNA fragment of appropriate length produced with the same restriction enzymecan be insertedinto the cut plasmid with DNA ligase.Plasmids containing a polylinker permit a researcher to use the same plasmid vector when cloning DNA fragmentsgenerated with different restriction enzymes,which simplifies experimentalprocedures. For some purposes,such as the isolation and manipulation of large segmentsof the human genome, it is desirable to clone DNA segments as large as several megabases [L megabase(Mb) : 1 million nucleotides).For this purpose specializedplasmid vectors known as BACs (bacterialartificial chromosomes)have been developed.One type of BAC uses a replication origin derived from an endogenousplasmid of E. coli known as the F factor. The F factor and cloning vectors derived from it can be stably maintained at a single copy per E. coli cell even when they contain inserted sequencesof up to about 2 Mb. Production of BAC libraries requires special methods for the isolation, ligation, and transformation of large segmentsof DNA becausesegments of DNA larger than about 20 kb are highly vulnerable to mechanical breakage by even standard manipulations such as prpetung.

DNA fragment to be cloned Enzymaticallyinsert DNA into plasmid vector

Recombinant plasmid

Mix E. coliwith olasmids in presenceol CaCl2;heat-pulse C u l t u r eo n n u t r i e n ta g a r p l a t e sc o n t a i n i n ga m p i c i l l i n

E. coli cnromosome

Transformedcell SUTVIVES

Cellsthat do not t a k e u p p l a s m i dd i e o n a m p i c i l l i np l a t e s

I P l a s m i replication d I

+

certmurtiotication I

g Colony of cells,each containingcopies of the same recombinantPlasmid

GDNALibrariesRepresentthe Sequences of Protein-CodingGenes A collection of DNA molecules each cloned into a vector molecule is known as a DNA library. !7hen genomic DNA from a particular organism is the source of the starting DNA, the set of clones that collectively represent all the DNA sequencesin the genome is known as a genomic D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N .

179

I lJ

+F*ry65' TranscriUe RNAintocDNA

< FIGURE 5-15 A cDNAlibrary containsrepresentative copiesof cellularmRNAsequences. A mixtureof mRNAsis pointfor preparing thestarting plasmid recombinant clones eachcontaining a cDNA.Transforming E coliwith the plasmids recombinant generates a setof cDNAclones representing allthecellular mRNAs. Seethetextfor a step-bysteodiscussion.

g I n " . o u " R N Aw i t ha t k a t i E J A d dp o t y ( d c ) t a i l Single-stranded 3' G GGGT-___--I cDNA

T T T T 5' t,,,.,.

EI lil$:fl6"#lli",

Y 5'I 3'GGGGI_---__--lTTTT5' -

complementary | Synttresize

sJ strand Double-stranded cDNA r (Jbbb a

-

i ^ F

3', - - r

I fTT5

oRl sites 5', 3'GGGG

E

I

C T T A A G E G G G G I _ _ _ _ _ _ - _ _T_ IT T T E C

lf Gf]GGGGI-.-_lT Stickyend

.

.,*"" withEcoRl T T TECTTAA

I Individual clones

180

T TAAGE

c H A p r E Rs

I

rJl:ru:HT:;,0?,,

MoLEcuLAR G E N E I Cr E c H N t e u E s

nJ.,,with EcoRl

library. Such genomic libraries are ideal for representingthe geneticcontent of relatively simple organismssuch as bacteria or yeast, but presentcertain experimental difficulties for higher eukaryotes.First, the genesfrom such organismsusually contain extensiveintron sequencesand therefore can be too large to be inserted intact into plasmid vectors. As a result, the sequencesof individual genesare broken apart and carried in more than one clone. Moreover, the presenceof introns and long intergenic regions in genomic DNA often makes it difficult to identify the important parts of a gene that actually encode protein sequences.For example, only about 1.5 percentof the hunran genomeactually represents protein-coding genesequences. Thus for many studies,cellular mRNAs, which lack the noncoding regionspresentin genomic DNA, are a more useful starting material for generating a DNA library. In this approach, DNA copies of mRNAs, called complementaryDNAs (cDNAs), are synthesized and cloned into plasmid vectors. A large collection of the resulting cDNA clones, representingall the mRNAs expressedin a cell type, is called a cDNA library.

cDNAsPreparedby ReverseTranscription o f C e l l u l a rm R N A sC a n B e C l o n e dt o G e n e r a t ec D N AL i b r a r i e s The first stepin preparing a cDNA library is to isolatethe total mRNA from the cell type or tissueof interest.Becauseof their poly(A) tails, mRNAs are easily separated from the much more prevalent rRNAs and tRNAs presentin a cell extract by use of a column to which short strings of thymidylate (oligo-dTs) are linked to the matrix. The generalprocedure for preparing a cDNA library from a mixture of cellular mRNAs is outlined in Figure 5-15. The enzymereversetranscriptase,which is found in retroviruses,is usedto synthesize a strand of DNA complementary to each mRNA molecule, starting from an oligo-dT primer (steps1 and2). The resulting cDNA-mRNA hybrid moleculesare converted in several stepsto double-strandedcDNA moleculescorrespondingto all the mRNA molecules in the original preparation (steps 3-5). Each double-stranded cDNA contains an oligodC.oligo-dG double-strandedregion at one end and an oligo-dT.oligo-dA double-strandedregion at the other end. Methylation of the cDNA protects it from subsequent restrictionenzymecleavage(step6). To prepare double-stranded cDNAs for cloning, short double-strandedDNA moleculescontaining the recognition site for a particular restriction enzymeare ligatedto both ends of the cDNAs using DNA ligasefrom bacteriophageT4 (Figure 5-15, step 7). As noted earlier,this ligasecan join "bluntended" double-strandedDNA moleculeslacking sticky ends. The resulting moleculesare then treated with the restriction enzymespecificfor the attachedlinker, generatingcDNA moleculeswith sticky endsat eachend (step8a). In a separateprocedure,plasmid DNA first is treatedwith the samerestriction enzymeto produce the appropriate sticky ends (step8b). The vector and the collection of cDNAs, all containing complementary sticky ends, then are mixed and joined covalently by DNA ligase(Figure 5-15, step 9). The resulting

DNA moleculesare transformed into E. coli cellsto generate individual clones;each clone carrying a cDNA derived from a singlemRNA. Becausedifferent genesare transcribed at very different rates,cDNA clonescorrespondingto abundantly transcribed genes will be representedmany times in a cDNA library, whereas cDNAs corresponding to infrequently transcribed geneswill be extremely rare or not presentat all. This property is advantageousif an investigatoris interestedin a gene that is transcribedat a high rate in a particular cell type' In this case,a cDNA library preparedfrom mRNAs expressedin that cell type will be enriched in the cDNA of interest, facilitating isolation of clonescarrying that cDNA from the library. Howeve! to have a reasonablechance of including clones corresponding to slowly transcribedgenes'mammalian cDNA libiaries must contain 1.06-107individual recombinantclones.

D N A L i b r a r i e sC a n B e S c r e e n e db y H y b r i d i z a t i o n e robe to an OligonucleotidP Both genomic and cDNA libraries of various organisms contain hundredsof thousandsto upwards of a million individual clones in the case of higher eukaryotes. Two general approaches are available for screening libraries to identify clonescarrying a geneor other DNA region of interest: (1) detectionwith oligonucleotideprobes that bind to the clone of interest and (2) detection basedon expression of the encoded protein. Here we describe the first method; an example of the secondmethod is presentedin the next section. The basis for screeningwith oligonucleotideprobes is hybridization, the ability of complementary singlestrandedDNA or RNA moleculesto associate(hybridize) specificallywith each other via basepairing. As discussed in Chapter 4, double-stranded(duplex) DNA can be denatured (melted) into single strands by heating in a dilute salt solution. If the temperature then is lowered and the ion concentration raised, complementary single strands will reassociate(hybridize) into duplexes.In a mixture of nucleic acids, only complementary single strands (or strands containing complementary regions) will reassociate; moreover,the extent of their reassociationis virtually unaffectedby the presenceof noncomplementarystrands. As we will seelater in this chapter,the ability to identify a particular DNA or RNA sequencewithin a highly complex mixture of moleculesthrough nucleic acid hybridization is the basis for many techniquesemployed to study gene expresslon. The stepsinvolved in screeningan E. coli plasmid cDNA library are depicted in Figure 5-16. First' the DNA to be screenedmust be attachedto a solid support. A replica of the petri dish containing a large number of individual E. coli clones is reproduced on the surfaceof a nitrocellulose membrane. The DNA on the membrane is denatured, and the membrane is then incubated in a solution containing a radioactively labeled probe specificfor the recombinant DNA containing the fragment of interest. Under hybridization conditions (near neutral pH, 40-65 "C' 0.3-0'6 M NaCl), D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N .

181

*'**

5eu

I n d i v i d u acl o l o n i e s

B o u n ds i n g l e - s t r a n d eDdN A

Master plate of E. coli colonies

Filter

P l a c en i t r o c e l l u l o sfei l t e ro n D l a t e t o p i c ku p c e l l sf r o m e a c hc o l o n y Hvbridized c o m p l e m e n t a rD y NAs

N i t r o c e l l u l o sfei l t e r

Wash away labeledDNA that does not hybridizeto DNA bound to filter

r"uorr autoradiography f

I

Performaut o r a d i o g r a p h y

o)

S i g n a la p p e a r so v e r p l a s m i dD N A t h a t i s complementary to probe

A EXPERIMENTAL FIGURE 5-16 cDNAlibrariescan be screened with a radiolabeledprobeto identifya cloneof interest.The a p p e a r a noc fea s p o to n t h ea u t o r a d i o g r ai nmd i c a t et hs ep r e s e n c e o f a r e c o m b i n acnl to n ec o n t a i n i nDgN Ac o m p l e m e n t a t or yt h e probeTheposition of the spoton the autoradioqram isthe mirror

i m a g eo f t h ep o s i t i oonf t h a tp a r t i c u l a c lro n eo n t h eo r i g i n aple t r i dish(although for easeof comparison, it isnot shownreversed h e r e )A. l i g n i ntgh ea u t o r a d i o g r awm i i s hw i l l i t ht h eo r i g i n aple t r d locatethe corresponding clonefromwhichE colicellscanbe recovered

this labeledprobe hybridizesro any complemenrarynucleic acid strandsbound to the membrane.Any excessprobe that doesnot hybridize is washedaway, and the labeledhybrids are detectedby autoradiography of the filter. This technique can be usedto screenboth genomicand cDNA libraries,but is most commonly usedto isolatespecificcDNAs. ClearlS identification of specific clones by the membrane-hybridizationtechnique depends on the availa b i l i t y o f c o m p l e m e n r a r yr a d i o l a b e l e d p r o b e s . F o r a n oligonucleotideto be useful as a probe, ir must be long enough for its sequenceto occur uniquely in the clone of interest and not in any other clones. For most purposes, this condition is satisfied by oligonucleotidescontaining a b o u t 2 0 n u c l e o t i d e s .T h i s i s b e c a u s ea s p e c i f i c2 0 - n u c l e o t i d e s e q u e n c eo c c u r s o n c e i n e v e r y 4 2 0 ( = 1 0 1 2 )n u cleotides.Since all genomesare much smaller (=3 x t0e nucleotidesfor humans),a specific20-nucleotidesequence in a genomeusually occurs only once. With automated ins t r u m e n t s n o w a v a i l a b l e , r e s e a r c h e r sc a n p r o g r a m t h e chemicalsynthesisof oligonucleotidesof specificsequence up to about 100 nucleotideslong. Longer probes can be preparedby the polymerasechain reaction (pCR), a widely

usedtechniquefor amplifying specificDNA sequences rhat is describedlater. How might an investigator design an oligonucleotide probe to identify a clone encoding a particular protein? It helps if all or a portion of the amino acid sequenceof the protein is known. Thanks to the availability of the complete genomic sequencesfor humans and some important model organisms such as the mouse, Drosophila, and the roundworm Caenorbabditis elegans,a researchercan use an appropriate computer program to searchthe genomic sequence database for the coding sequencethat corresponds to the amino acid sequenceof the protein under study. If a match is found, then a single, unique DNA probe based on this known genomic sequencewill hybridize perfectly with the clone encoding the protein of interest.

182

.

cHAprER s

I

M o l E c u L A RG E N E T rI cE c H N t e u E S

YeastGenomicLibrariesCan Be Constructed with Shuttle Vectorsand Screenedby F u n c t i o n aC l omplementation In somecasesa DNA library can be screenedfor the ability to express a functional protein that complements a recessive

Polylinker

(a)

Shuttlevector

CEN (b)

l^

dNos mil$

mmrs

Yeastgenomic DNA

Transform E. coli Screenfor amoicillinresistance 'as\ +

*

***

I lsolateandpoolrecombinant from 105transformed I plasmids { F. colicolonies complementation Assayyeastgenomiclibraryby functional

5-17 A yeastgenomiclibrarycan be < EXPERIMENTAL FIGURE plasmid vector that can replicatein shuttle in a constructed shuttlevector of a typicalplasmid yeastand E.coli.(a)Components genesThepresence of a yeastoriginof for cloningSaccharomyces (CEN) (ARS) allowsstable anda yeastcentromere DNAreplication isa yeast yeast included Also in andsegregation replication markersuchasURA3,whichallowsa ura3 mutantto selectable sequences thevectorcontains growon mediumlackinguracilFinally, in E.coli(ORlandamp')anda polylinker andselection for replication (b)Typical protocol for of yeastDNAfragments. for easyinsertion yeast of total digestion genomic Partial yeast library a constructing with an fragments to generate genomic DNAwith5au3Aisadjusted to acceptthe sizeof about10 kb Thevectorisprepared average the whichproduces with BamHl, genomic by digestion fragments E. coli that clone of transformed samestickyendsas5au3A.Each a singletype contains resistance for ampicillin growsafterselection of yeastDNAfragment in a genomic DNA fragment inserted into a plasmid vector. To construct a plasmid genomic library that is to be screenedby functional complementationin yeast cells,the plasmid vector must be capable of replication in both E. coli cells and yeast cells. This type of vector' capable of propagation in two different hosts, is called a shuttle vector. The structure of a typical yeast shuttle vector is shown in Figure 5-L7a. This vector contains the basic elements that permit cloning of DNA fragmentsin E- coli.In addition, the shuttle vector contains an autonomouslyreplicating sequence(ARS), which functions as an origin for DNA replication in yeast; a yeast centromere (called CEN), which allows faithful segregationof the plasmid during yeastcell division; and a yeastgeneencodingan enzymefor uracil synthesis (URA3), which serves as a selectable marker in an approprlate yeastmutant. To increasethe probability that all regions of the yeast genome are successfullycloned and represented in the plasmid library, the genomic DNA usually is only partially digest.d to yield overlapping restriction fragments of =tb t U. These fragments are then ligated into the shuttle vector in which the polylinker has been cleavedwith a restriction enzymethat produces sticky ends complementary to those on the yeast DNA fragments (Figure 5-17b). Becausethe 10-kb restriction fragmentsof yeastDNA are incorporated into the shuttle u.ito., randomly, at least 10s E. coli colonies,each containing a particular recombinant shuttle vector, are necessaryto assurethat each region of yeast DNA has a high probability of being representedin

the library at least once. mutation. Sucha screeningstrategywould be an efficientway Figure 5-18 outlines how such a yeast genomic library to isolate a cloned genethat correspondsto an interestingrecan be screenedto isolate the wild-type gene corresponding cessivemutation identified in an experimental organism. To one of the temperature-sensitivecdc mutations mentioned to illustrate this method, referredto as functional complementain this chapter. The starting yeast strain is a double earlier E. coli tion, we describehow yeast genescloned in special that requires uracil for growth due to a ura3 mttamutant identify yeast cells to plasmidscan be introduced into mutant is temperature-sensitivedue to a cdc28 mutation and tion strain. in the mutant the wild-type genethat is defective by its phenotype (see Figure 5-6). Recombinant identified purpose of screening for the Libraries constructed from the yeast genomic library are mixed isolated plasmids from constructed are among yeast gene sequencesusually genomic DNA rather than cDNA. BecauseSaccharomyces with yeast cells under conditions that promote transformation of the cells with foreign DNA. Sincetransformed yeast genesdo not contain multiple introns, they are sufficiently cells carry a plasmid-borne copy of the wild-type URA3 compact that the entire sequenceof a genecan be included D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N

183

Libraryof yeast genomic DNA carrying URA3selectivemarker

Temperature-sensitive cdc-mutant yeast; ura3 (requiresuracil)

Transformyeast by treatmentwith LiOAC,PEG.and heat shocr Plateand incubateat permissivetemperature o n m e d i u m l a c k i n gu r a c i l

Only colonies carrYtng a URA3 marker are able to grow

23'C

R e p l i c a - p l a taen d incubateat nonpermissive te m perature

Only colonies carrying a wild-type CDC gene are able to grow

36'C

EXPERIMENTAL FIGURE 5-18 Screening of a yeastgenomic libraryby functionalcomplementation can identifyclones carryingthe normalform of a mutantyeastgene.Inthis example, a wild-type CDCgeneis isolated by complementation of a cdcyeastmutant TheSaccharomyces strainusedfor screentng rne yeastlibrary carries ura3 anda temperature-sensitive cdcmutation Thismutantstrainisgrownandmaintained at a permisstve (23"C) Pooled temperature plasmids prepared recombinant as

shownin Figure 5-17arerncubated with the mutantyeastcellsunder conditions thatpromotetransformation Therelatively few transformed yeastcells, plasmid whichcontainrecombinant DNA. cangrowin theabsence of uracilat 23 'C Whentransformed yeast colonies arereplica-plated andplacedat 36'C (a nonpermissrve temperature), onlyclones carrying a libraryplasmid thatcontains the wild-type copyof the CDCgenewillsurviveL|OAC= lithiumacetate; PEG= polyethylene glycol

gene,they can be selectedby their ability to grow in the absenceof uracil. Typically, about 20 petri dishes, each containing about 500 yeast transformants, are sufficient to represent the entire yeast genome. This collection of yeast transformants can be maintained at 23"C, a temperature permissivefor growth of the cdc28 mutant. The entire colIection on 20 plates is then transferredto replica plates, which are placed at 36 "C, a nonpermissivetemperature for cdc mutants. Yeastcoloniesthat carry recombinant plasmids expressinga wild-type copy of the CDC28 genewill be able to grow at 36'C. Once temperature-resistant yeastcolonies have been identified, plasmid DNA can be extracted from the cultured yeast cells and analyzed by subcloning and DNA sequencing,topics we take up next.

the well into which the original DNA mixture was placed at the start of the electrophoreticrun. Smaller moleculesmove through the gel matrix more readily than larger molecules, so that molecules of different length migrate as distinct bands. Smaller DNA moleculesfrom about 10 to 2000 nucleotides can be separatedelectrophoretically on polyacrylamide gels, and larger molecules from about 200 nucleotidesto more than 20 kb on agarose gels. A common method for visualizingseparatedDNA bands on a gel is to incubatethe gel in a solution containing the fluorescentdye ethidium bromide. This planar moleculebinds to DNA by intercalating berweenthe base pairs. Binding concentratesethidium in the DNA and also increasesits intrinsic fluorescence.As a result, when the gel is illuminated with ultraviolet light, the regions of the gel containing DNA fluoresce much more brightly than the regionsof the gel without DNA. Once a cloned DNA fragment, especiallya long one, has beenseparatedfrom vector DNA, it often is treated with various restriction enzymesto yield smallerfragments.After separation by gel electrophoresis,all or some of these smaller fragments can be ligated individually into a plasmid vector and cloned in E. coli by the usual procedure. This process, known as subcloning,is an important step in rearranging parts of genesinto useful new configurations. For instance, an investigator who wants to change the conditions under which a geneis expressedmight usesubcloningto replacethe normal promoter associatedwith a cloned genewith a DNA segmentcontaining a different promoter. Subcloningalso can be used to obtain cloned DNA fragments that are of an appropriate length for determining the nucleotidesequence.

Gel Electrophoresis Allows Separationof Vector DNA from ClonedFragments In order to manipulate or sequencea cloned DNA fragment, it sometimesmust first be separatedfrom the vector DNA. This can be accomplished by cutting the recombinant DNA clone with the samerestriction enzymeusedto produce the recombinant vectors originally. The cloned DNA and vector DNA then are subjected to gel electrophoresis, a powerful method for separatingDNA moleculesof different size(Figure5-19). Near neutral pH, DNA molecules carry a large negative charge and therefore move toward the positive electrode during gel electrophoresis.Becausethe gel marrix restricrs random diffusion of the molecules, molecules of the same length migrate together as a band whose width equalsthat of '184 .

c H A p r E5 R | M o L E c u L AGRE N E TrtEc c H N t e u E s

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dKs

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separates 5-19 Gel electrophoresis FIGURE < EXPERIMENTAL by DNA moleculesof different lengths.(a)A gel is prepared p o u r i n ga l i q u i dc o n t a i n i negi t h e rm e l t e da g a r o soer t w o g l a s sp l a t e sa f e w u n p o l y m e r i zaecdr y l a m i dbee t w e e n m i l l i m e t ear sp a r t A s t h e a g a r o sseo l i d i f i eosr t h e a c r y l a m i d e e v a l sf)o r m s p o l y m e r i z ienst op o l y a c r y l a m i ad eg ,e lm a t r i x( o r a n g o p o l y m e r s T h e d i m e n s i o nosf o f c h a i n s c o n s i s t i nogf l o n g ,t a n g l e d s ,r p o r e sd, e p e n do n t h e t h e i n t e r c o n n e c t icnhga n n e l o f t h e a g a r o soer a c r y l a m i dues e dt o f o r mt h e g e l concentration T h es e p a r a t ebda n d sc a nb e v i s u a l i z ebdy a u t o r a d i o g r a p(hi fyt h e n yt e f r a g m e n tasr er a d i o l a b e l eodr)b y a d d i t i o no f a f l u o r e s c e d ( e g , e t h i d i u mb r o m i d et )h a t b i n d st o D N A ( b )A p h o t o g r a pohf a ( E t B r )E t B rb i n d st o D N Aa n d g e ls t a i n e w d i t h e t h i d i u mb r o m i d e f l u o r e s c eusn d e rU Vl i g h t ,T h eb a n d si n t h ef a r l e f ta n df a r r i g h t f r a g m e n tosf k n o w ns i z e l a n e sa r ek n o w na s D N Al a d d e r s - D N A h e l e n g t ho f t h e D N A f o r d e t e r m i n i nt g t h a ts e r v ea sa r e f e r e n c e f r a g m e n tisn t h e e x p e r i m e n tsaal m p l eI P a r(tb )S c i e n cPeh o t o L i b r alr y

o- o - PI : o

-O-P:O

I o

I

o

- O - P I: O

- O - P I: O

I o Subjectto autoradiography or incubatewith fluorescentdye

I

-O-P:O

l o

ase

- O - PI : O I O

I

CH,

QH,

HH Signals corresponding to DNA bands

Deoxyribonucleoside triphosPhate (dNTP)

Dideoxyribonucleoside triphosPhate (ddNTP)

of deoxyribonucleoside 5-20 Structures FIGURE triphosphate (dNTP) dideoxyribonucleoside and triphosphate intoa growingDNA residue (ddNTP). of a ddNTP Incorporation at thatpoint elongation strandterminates D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N

185

Technique Animation:DideoxySequencing of DNA{tttt (b)

P r i m e r5 ' T e m p l a t e3 ' -

5'

(a) 5'TAGCTGACTC3' 3' A T C G A C T G A G T C A A G A A C T A T T G G G C T T A A

I DNApolymerase

+ ddGTP

I | + dATB dGTB dCTB dTTp | + ddGTPin low concentration I

v 5'TAGCTGACTCAG3' 3' ATCGACTGAGTCAAGAACTATTGGGCTTAA

-A

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5 ' T A G C T G A CT C A GT T C + T T G 3 ' 3' ATCGACTGAGTCAAGAACTATTGGGCTTAA

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5' TAGCTGACTCAGTTJTTCNTNACCCG3' 3' ATCGACTGAGTCAAGAACTATTGGGCTTAA

-T

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etc.

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etc.

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Denatureand separatedaughterstrandsby electrophoresis I N N N N A A T G A A TA G A T A T A T A G G G G A A T T G A G T 20 30 40

AAATAG 110

TTGG GTAA 120

ATGGT 130

GGTA

ATAG

GGGGAT

TGTTT 140

a EXPERIMENTAL FTGURE 5-21 ClonedDNAscan be sequencedby the Sangermethod, usingfluorescent-tagged dideoxyribonucleoside (ddNTps). triphosphates (a)A single (template) strandof the DNAto be sequenced (blueletters) is hybridized to a synthetic primer(blackletters) deoxyribonucleotide Theprimeriselongated in a reaction mixture containing thefour normaldeoxyribonucleoside triphosphates plusa relatively small amountof oneof thefourdideoxyribonucleoside triphosphates. ln thisexample, (yellow) ddGTP ispresentBecause of the relatively low concentration of ddGTP, incorporation of a ddGTBandthus chaintermination, occurs at a givenposition in the sequence only about1 percent of thetime Eventually the reaction mixture will containa mixture of prematurely (truncated) terminated dauqhter

186

CHAPTER 5

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MOLECULAG RENETIC TECHNIQUES

T

TAGAGT

GA

TG

AGG

GTGAAATTGTTATGTAAA 150 160

AIG 80

AAG TTGAGTATT 90-

170

A

A

AA '180

I

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fragments endingat everyoccurrence of ddGTp(b)Toobtainthe complete sequence of a templateDNA,four separate reactions are performed, eachwith a different dideoxyribonucleoside triphosphate (ddNTP) TheddNTP thatterminates eachtruncated fragment canbe identified by useof ddNTPs taggedwith four differentfluorescent dyes(indicated (c)In an automated bycolored highlights). sequencing machine, thefourreaction mixtures aresubjected to gel electrophoresis, andtheorderof appearance of eachof thefour drfferent fluorescent dyesat the endof the gelis recordedShown hereisa sample printoutfroman automated sequencer fromwhich thesequence of theoriginal template DNAcanbe deduced fromthe sequence of thesynthesized strandN = nucleotide thatcannotbe (c)fromGriffiths assigned[Part et al, Figure 14-27 I

lsolation of a genomic library spanning the genome of interest A l i g n i n gl i b r a r yc l o n e s by hybridizationor restriction-site mapping

Ordered set of clones spanningthe genome

Sequencing of orderedclones

Genomicsequence > FIGURE 5-22 Two Strategies for Assembling WholeGenome Sequences. Onemethoddepends on isolating andassembling a set thatspanthe genomeThiscanbe doneby of clonedDNAsegments or by alignment of matching clonedsegments by hybridization restriction sitemapsTheDNAsequence of the ordered clones can genomic intoa complete The thenbe assembled sequence

of Sequencing r a n d o ml i b r a r yc l o n e s

Sequenceof unordered fragments, for about lO-fold coverageof eachgenomic segment

Aligning sequenced clonesby comPuter Assembled genomlcsequence DNA easeof automated on the relative methoddepends alternative the library stepof ordering the laborrous andbypasses sequencing sothateachsegment clones enoughrandomlibrary Bysequencing to it ispossible to 10 times genome from 3 is represented of the of the alignment by computer sequence thegenomrc reconstruct fragments verylargenumberof sequence

guished by their corresponding fluorescent label (Figure 521b). For example,all truncated fragmentsthat end with a G would fluoresce one color (e.g., yellow), and those ending with an A would fluoresceanother color (e.g',red), regardless The complete characterizationof any cloned DNA fragment requires determination of its nucleotide sequence.F. Sanger of their lengths. The mixtures of truncated daughter fragments from each of the four reactionsare subjectedto elecand his colleaguesdeveloped the method now most comtrophoresison specialpolyacrylamidegels that can separate monly used to determine the exact nucleotide sequenceof single-strandedDNA moleculesdiffering in length by only 1 DNA fragmentsup to =500 nucleotideslong. The basic idea nucleotide.A fluorescencedetector that can distinguish the behindthis method is to synthesizefromthe DNA fragmentto four fluorescenttags is located at the end of the gel. The sebe sequenceda set of daughterstrandsthat are labeledat one quence of the original DNA template strand can be deterend and differ in length by one nucleotide.Separationof the mined from the order in which different labeledfragmentsmitruncated daughterstrandsby gel electrophoresiscan then esgrate past the fluorescencedetector (Figure 5-21'c). tablish the nucleotidesequenceof the original DNA fragment. In order to sequencea long continuousregion of genomic Synthesisof truncated daughterstandsis accomplishedby triphosphates(ddNTPs). DNA or even the entire genomeof an organism, researchers use of 2',3'-dideoxyribonucleoside These molecules,in contrast to normal deoxyribonucleotides usually employ one of the strategiesoutlined in Figure 5-22. (dNTPs), lack a 3' hydroxyl group (Figure 5-20). Although The first method requiresthe isolation of a collection of cloned overlap. Once the sequence DNA fragmentswhose sequences ddNTPs can be incorporated into a growing DNA chain by oligonucleotidesbased is determined, fragments of these of one DNA polymerase,once incorporated they cannot form a for use as synthesized can be chemically sequence on that phosphodiesterbond with the next incoming nucleotide In fragments. overlapping the adjacent in sequencing primers triphosphate.Thus incorporation of a ddNTP terminates is determined DNA of long stretch of a sequence way, the this resulting at in a daughterstrand truncated chain synthesis, incrementally by sequencingof the overlapping cloned DNA specific positions correspondingto the basecomplementary fragments that compose it. A second method, which is called added ddNTP on the template strand. to the the time-consumbypasses whole genomeshotgunseqwencing, Sequencingusing the Sangerdideoxy chain-termination ing step of isolating an ordered collection of DNA segments method is usually carried out using an automated DNA sethat span the genome.This method involves simply sequencing quencing machine. The reaction begins by denaturing a random clones from a genomic library. A total number of double-strandedDNA fragment to generatetemplate strands for in vitro DNA synthesis.A syntheticoligodeoxynucleotide clonesare chosenfor sequencingso that on averageeach segabout 10 times.This degreeof ment of the genomeis sequenced is usedas the primer for the polymerizationreactionthat concoverageensuresthat eachsegmentof the genomeis sequenced tains a low concentrationof eachof the four ddNTPs in addimore than once.The entiregenomicsequenceis then assembled tion to higher concentrationsof the normal dNTPs. The using using a computeralgorithm that alignsall the sequences, ddNTPs are randomly incorporated at the positions of the their regionsof overlap.\7hole genomeshotgunsequencingis correspondingdNTP, causingtermination of polymerization of the fastestand most cost-effectivemethod for sequencinglong at thosepositionsin the sequence(Figure5-21,a).Inclusion stretchesof DNA, and most genomes'including the human fluorescenttagsof different colors on eachof the four ddNTPs genome,have beensequencedby this method. allows each set of truncated daughterfragmentsto be distin-

Rapidly C l o n e dD N A M o l e c u l e sA r e S e q u e n c e d b y t h e D i d e o x yC h a i n - T e r m i n a t i oMne t h o d

D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N

187

TechniqueAnimation: PolymeraseChain Reaction{tttt

c Y c l e1

, Denaturationof DNA I A n n e a l i n go f p r i m e r s

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c Y c l e2

, Denaturationof DNA I A n n e a l i n go f p r i m e r s

Elongationof primers

{

G Y c l e3

< EXPERIMENTAL FIGURE 5-23 The polymerase chainreaction(PCR) is widely used to amplifyDNAregionsof known sequences. Toamplifya specific regionof DNA,an investigator willchemically primers synthesize two different oligonucleotrde complementary to (designated sequences of approximately 18 bases flankingtheregionof interest aslightblue anddarkbluebars)Thecomplete reaction iscomposed of a complex mixture of doublegenomic stranded DNA(usually DNAcontaining thetargetsequence of interest), a stoichiometric excess of bothprimers, thefourdeoxynucleoside triphosphates, anda heatstableDNApolymerase knownasTaqpolymerase. DuringeachPCRcycle, the reaction mixture isfirstheatedto separate thestrands andthencooledto allowtheprimers to bindto complementary sequences flanking the regionto beamplified. Iaq polymerase thenextends eachprimerfromits3' end,generating newlysynthesized strands thatextendin the3' direction to the 5' endof thetemplate strand.Duringthethirdcycle, two double-stranded DNAmolecules aregenerated equalin lengthto thesequence of theregionto beamplified Ineachsuccessive cyclethetargetsegment, whichwillannealto theprimers, isduplicated, andwilleventually vastlyoutnumber allotherDNAsegments in thereaction mixture Successive PCRcycles canbeautomated bycycling the reaction for timedintervals at high temperature for DNAmeltingandat a definedlowertemperature for theannealing and portions elongation of thecycleA reaction thatcycles 20 timeswillamplifythespecific target sequence 1-million-fold

, Denaturationof DNA I n n n e a t i n so f p r i m e r s

E l o n g a t i o no f p r i m e r s

I

L

Cycles4, 5, 6, etc.

T h e P o l y m e r a sC e h a i nR e a c t i o nA m p l i f i e sa SpecificDNA Sequencefrom a ComplexMixture If the nucleotide sequencesat the ends of a parricular DNA region are known, the intervening fragment can be amplified directly by the polymerase chain reaction (PCR). Here we describethe basic PCR technique and three situations in which it is used. 188

.

cHAprER s

I

M o L E c u L AG R E N E I cr E c H N t e u E s

The PCR dependson the ability to alternately denature (melt) double-strandedDNA moleculesand hybridizecomplementary singlestrandsin a controlled fashion. As outlined in Figure5-23, a typical PCR procedurebeginsby heat-denaturation of a DNA sampleinto singlestrands.Next, two synthetic oligonucleotidescomplementaryto the 3' ends of the target DNA segmentof interestare addedin greatexcessto the denatured DNA, and the temperarure is lowered to 50-60 'C. Thesespecificoligonucleotides,which are at a very high concentration,will hybridizewith their complementarysequences in the DNA sample, whereas the long strands of the sample DNA remain apart becauseof their low concentration.The hybridized oligonucleotidesthen serveas primers for DNA chain synthesisin the presenceof deoxynucleotides(dNTPs) and a temperature-resistantDNA polymerase such as that from Thermusaquaticus(a bacteriumthat livesin hot springs).This enzymq called Taq polymerase, can remain active even after being heatedto 95 oC and can extend the primers at temperaturesup to72"C. When synthesisis complete,the whole mixture is then heatedto 95 "C to denaturethe newly formed DNA duplexes.After the temperatureis lowered again, another cycle of synthesistakes place becauseexcessprimer is still present. Repeated cycles of denaturation (heating) followed by hybridization and synthesis(cooling) quickly amplify the sequence of interest. At each cycle, the number of copies of the sequencebetweenthe primer sitesis doubled; therefore, the desired sequenceincreasesexponentially-about a million-fold after 20 cycles-whereas all other sequencesin the original DNA sampleremain unamplified. Direct lsolation of a Specific Segment of Genomic DNA For organismsin which all or most of the genome has been sequenced,PCR amplification starting with the total genomic DNA often is the easiestway to obtain a specificDNA region of interest for cloning. In this application, the two oligonucleotideprimers are designedto hybridize to sequences flanking the genomic region of interest and to include sequences

Regionto be amplified 5'

3'

P r i m e r1

D N As y n t h e s i s

s'

5'

P r i m e r2

5-24 A specifictarget region in total FIGURE < EXPERIMENTAL genomicDNAcan be amplifiedby PCRfor usein cloning.Each to oneendof thetargetsequence primerfor PCRiscomplementary that enzyme for a restriction sequence the recognition andincludes primer doesnot havea sitewithinthetargetregion.In thisexample, primer2 contains a Hindlll whereas a BamHl sequence, 1 contains (Notethatfor clarity, icationof only in anyround,amplif sequence isshown,the onein brackets oneof thetwo strands ) After aretreatedwith appropriate thetargetsegments amplification, generating with stickyendsThese fragments restriction enzymes, plasmid vectors andcloned into complementary canbe incorporated (seeFigure 5-13). in E colibythe usualprocedure

c

Prime1 r

C o n t i n u ef o r = 2 0 PCRcycles Cut with restriction enzymes

Stickyend

-Stickyend Ligatewith plasmidvector with stickyends

that are recognizedby specificrestriction enzymes(Figure 524). After amplification of the desired target sequencefor about 20 PCR cycles,cleavagewith the appropriate restriction enzymesproduces sticky ends that allow efficient ligation of the fragment into a plasmid vector cleaved by the same restriction enzymesin the polylinker. The resulting recombinant plasmids,all carrying the identical genomic DNA segment,can then be cloned in E. coli cells.\fith certain refinements of the PCR, even DNA segmentsgreater than 10 kb in length can be amplified and cloned in this way. Note that this method does not involve cloning of large numbers of restriction fragments derived from genomic DNA and their subsequentscreeningto identify the specificfragment of interest. In effect, the PCR method inverts this traditional approach and thus avoids its most tedious aspects.The PCR method is useful for isolating genesequencesto be manipulated in a variety of useful ways describedlater. In addition the PCR method can be used to isolate gene sequencesfrom mutant organismsto determinehow they differ from the wild type. A variation on the PCR method allows PCR amplification of a specificcDNA sequencefrom cellular mRNAs. This

method, known as reuersetranscriptase-PcR /R?PCR/, begins with the same procedure describedpreviously for isolation of cDNA from a collection of cellular mRNAs' Typically' an oligo-dT primer, which will hybridize to the 3' poly(A) tail of the mRNA, is used as the primer for the first strand of cDNA synthesisby reversetranscriptase.A specific cDNA can then be isolatedfrom this complex mixture of cDNAs by PCR amplification using two oligonucleotide primers designedto match sequencesat the 5' and 3' ends of the corresponding mRNA. As described previously, these primers could be designedto include restriction sitesto facilitate the insertion of amplified cDNA into a suitableplasmid vector. Preparation of Probes Earlier we discussedhow oligonucleotide probes for hybridization assayscan be chemically synthesized.Preparation of such probes by PCR amplification requires chemical synthesisof only two relatively short primers corresponding to the two ends of the target sequence. The starting sample for PCR amplification of the target sequencecan be a preparation of genomic DNA, or a preparation of cDNA synthesizedfrom the total cellular 32PmRNA. To generatea radiolabeled product from PCR, labeled dNTPs are included during the last several amplification cycles.Becauseprobes prepared by PCR are relatively 3'P atoms incorporated into long and have many radioactive them, theseprobes usually give a stronger and more specific signal than chemically synthesizedprobes. Tagging of Genes by Insertion Mutations Another useful application of the PCR is to amplify a "tagged" gene from the genomic DNA of a mutant strain. This approach is a simpler method for identifying genesassociatedwith a particular mutant phenotype than screening of a library by functionalcomplementation(seeFigure5-18). The key to this use of the PCR is the ability to produce mutations by insertion of a known DNA sequenceinto the genome of an experimental organism. Such insertion mutations can be generated by use of mobile DNA elements, which can move (or transpose)from one chromosomal site to another. As discussedin more detail in Chapter 5, these DNA sequencesoccur naturally in the genomesof most organisms and may give rise to loss-of-function mutations if they transposeinto a protein-coding region. For example, researchershave modified a Drosophila mobile DNA element, known as the P element, to optimize D N A C L O N I N GA N D C H A R A C T E R I Z A T I O N

r89

> EXPERIMENTAL FIGURE 5-25 The genomic sequenceat the insertionsite of a transposon is revealedby PCRamplificationand Restriction sites:t sequencing. Toobtainthe DNAsequence of the insertion siteof a P-element transooson it is necessary to PCR-amplify thejunctionbetween knowntransposon sequences andunknown flankingchromosomal sequences Onemethodto achieve thisisto cleave genomic DNAwith a restriction enzyme thatcleaves oncewithinthe transposon sequence Ligation of the resulting restriction fragments willgenerate circular DNA molecules. Byusingappropriately designed DNA primers thatmatchtransposon sequences it is possible junction to PCR-amplify the desired fragmentFinally, (see a DNAsequencing reaction Figure 5-21)is performed usrngthe PCR-amplified fragmentasa template andan oligonucleotide primerthat matches sequences neartheendof the transposon, to obtainthesequence of thejunction between thetransposon andchromosome

I ransDoson

I cr, *it, I

restriction enzyme

Ligate to circularize

PCRprimers

Sequencing pflmer

I PCRamplification with Orimers to transposon I

-+

its use in the experimental generation of insertron mutations. Once it has been demonstratedthat insertion of a P element causesa mutation with an interestingphenotype, the genomic sequencesadjacentto the insertion site can be amplified by a variation of the standardPCR protocol that usessyntheticprimers complementaryto the known P-element sequencebut that allows unknown neighboring sequencesto be amplified. One such method, depicted in Figure 5-25, beginsby cleavingDrosophila genomic DNA containing a P-elementinsertion with a restriction enzyme that cleavesonce within the P-elementDNA. The collection of cleaved DNA fragmenrs treated with DNA ligase yields circular molecules,some of which will contain P-element DNA. The chromosomal region flanking the P element can then be amplified by PCR using primers that match P-elementsequencesand are elongatedin opposite directions. The sequenceof the resulting amplified fragment can then be determined using a third DNA primer. The crucial sequencefor identifying the site of P-element insertion is the junction between the end of the P-element and genomic sequences.Overall, this approach avoids the cloning of large numbers of DNA fragments and their screeningto detect a cloned DNA correspondingto a mutated geneof interest. Similar methods have been applied to other organrsms for which insertion mutations can be generatedusing either mobile DNA elements or viruses with sequencedgenomes that can insertrandomly into the genome. 190

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DNA Cloning and Characterization r In DNA cloning, recombinant DNA molecules are formed in vitro by inserting DNA fragments into vecror DNA molecules.The recombinant DNA molecules are then introduced into host cells, where they replicate, producing large numbers of recombinant DNA molecules. r Restriction enzymes (endonucleases)typically cut DNA at specific4- to 8-bp palindromic sequences,producing defined fragments that often have self-complementarysinglestrandedtails (stickyends). I Two restriction fragments with complementary ends can be joined with DNA ligase to form a recombinant DNA molecule(seeFigure 5-12). t E. coli cloning vectors are small circular DNA molecules (plasmids)that include three functional regions:an origin of replication, a drug-resistancegene, and a site where a DNA fragmentcan be inserted.Transformedcellscarrying a vector grow into colonieson the selectionmedium (seeFigure5-13). r A cDNA library is a set of cDNA clones prepared from the mRNAs isolated from a particular type of tissue. A genomic library is a set of clones carrying restriction fragments produced by cleavageof the entire genome. r In cDNA cloning, expressed mRNAs are reversetranscribedinto complementaryDNAs, or cDNAs. By a series of reactions,single-strandedcDNAs are converted into

double-strandedDNAs, which can then be ligated into a plasmidvector (seeFigure5-15). r A particular cloned DNA fragment within a library can be detected by hybridization to a radiolabeledoligonucleotidewhose sequenceis complementaryto a portion of the fragment(seeFigure5-16). r Shuttle vectors that replicate in both yeast and E. coli can be used to construct a yeastgenomic library. Specificgenes can be isolated by their ability to complementthe correspondingmutant genesin yeastcells(seeFigure5-17). r Long cloned DNA fragments often are cleavedwith restriction enzymes,producing smallerfragmentsthat are then separatedby gel electrophoresisand subclonedin plasmid vectorsprior to sequencingor experimentalmanipulation. r DNA fragmentsup to about 500 nucleotideslong are sequenced in automated instruments based on the Sanger (dideoxychain-termination)method (seeFigure5-21). r lil/hole genome sequencescan be assembledfrom the sequencesof a large number of overlappingclones from a genomiclibrary (seeFigure 5-22). r The polymerasechain reaction(PCR)permitsexponential amplification of a specificsegmentof DNA from just a single initial template DNA molecule if the sequenceflanking the DNA regionto be amplifiedis known (seeFigure5-23). r PCR is a highly versatilemethod that can be programmed to amplify a specificgenomicDNA sequence,a cDNA, or a sequenceat the junction betweena transposableelement and flanking chromosomalsequences.

EE UsingClonedDNAFragments to StudyGeneExpression In the last sectionwe describedthe basictechniquesfor using recombinantDNA technologyto isolatespecificDNA clones, and ways in which the clones can be further characterized.

Now we considerhow an isolatedDNA clone can be usedto study geneexpression.We discussseveralwidely usedgeneral techniquesthat rely on nucleic acid hybridization to elucidate when and where genesare expressed,as well as methods for generatinglarge quantitiesof protein and otherwisemaniputo determinetheir expressionpatlating amino acid sequences terns,structure,and function. More specificapplicationsof all thesebasictechniquesare examinedin the following sections.

s e r m i tD e t e c t i o no f H y b r i d i z a t i o nT e c h n i q u eP F r a g m e n t s a n d mRNAs S p e c i f i cD N A Two very sensitivemethodsfor detectinga particular DNA or RNA sequencewithin a complex mixture combine separation and hybridizationwith a complementary by gel electrophoresis probe. A third method involveshybridizing DNA radiolabeled labeledprobesdirectly onto a preparedtissuesample.\Wewill encounter referencesto all three of these techniques,which have numerousapplications,in other chapters. Southern Blotting The first hybridizationtechniqueto detect DNA fragments of a specific sequenceis known as Southern blotting after its originator E. M. Southern.This techniqueis capable of detecting a single specific restriction fragment in the highly complex mixture of fragments produced by cleavageof the entire human genomewith a restrictionenzyme.When sucha complex mixture is subjectedto gel electrophoresis,so many different fragmentsof nearly the same length are presentit is not possibleto resolveany particular DNA fragmentsas a discrete band on the gel. Neverthelessit is possibleto identify a particular fragmentmigrating as a band on the gel by its ability to hybridize to a specificDNA probe. To accomplish this, the restriction fragments present in the gel are denatured with alkali and transferredonto a nitrocellulosefilter or nylon membraneby blotting (Figure 5-26). This procedurepreservesthe distribution of the fragmentsin the gel, creatinga replica of the gel on the filter. (The blot is usedbecauseprobesdo not readily diffuseinto the original gel.)The filter then is incubatedunder hybridization conditions with a specificradiolabeledDNA probe, which usually is

DNA I I Cleavewith restriction enzymes I Gel V

Autoradiogram

Ni t r o c e l l luo s e Ni t r o c e l l luo s e Gel ----.f----

Hybridizewith l a b e l e dD N A o r R N Ap r o b e

---r---T---T

A l k a l i n es o l u t i o n C a p i l l a r ya c t i o nt r a n s f e r s DNAfrom gelto nitrocellulose

FIGURE A EXPERIMENTAL 5-25 Southernblot techniquecan detecta specificDNAfragmentin a complexmixtureof threedifferent restrictionfragments.Thediagram depicts to fragments in the gel,butthe procedure canbe applied restriction

that of DNAfragmentsOnlyfragments of millions a mixture probewillgivea signalon an autoradiogram to a labeled hybridize mRNAs specifrc blottingdetects calledtVorthern technique A similar J Mol Blol98:508 1975, l withina mixturelseeE M Southern,

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generatedfrom a cloned restriction fragment. The DNA restriction fragmentthat is complementaryto the probe hybridizes,and its location on the filter can be revealedby autoradiography. Northern Blotting One of the most basicways to characterize a cloned geneis to determinewhen and where rn an organism the geneis expressed.Expressionof a particular genecan be followed by assayingfor the corresponding mRNA by Northern blotting, named, in a play on words, after the related method of Southern blotting. An RNA sample, often the total cellular RNA, is denatured by treatment with an agent such as formaldehyde that disrupts the hydrogen bonds between base pairs, ensuring that all the RNA moleculeshave an unfolded, linear conformation. The individual RNAs are separated according to size by gel electrophoresisand transferred to a nitrocellulose filter to which the extendeddenatured RNAs adhere.As in Southernbloning, the filter then is exposedto a labeled DNA probe that is complementaryto the geneof interest; finally, the labeled filter is subjected to autoradiography. Becausethe amount of a specificRNA in a samplecan be estimated from a Norrhern blot, the procedureis widely used ro compare the amounts of a particular mRNA in cells under differentconditions(Figure5-27). In Situ Hybridization Northern blotting requiresextracring the mRNA from a cell or mixture of cells,which meansthat the cellsare removedfrom their normal location within an orsan-

UN

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ism or tissue.As a result, the location of a cell and its relation to its neighbors is lost. To retain such positional information in precisestudiesof geneexpression,a whole or sectionedtissueor evena whole permeabilizedembryo may be subjectedto in situ hybridization to detect the mRNA encoded by a particular gene.This technique allows genetranscription to be monitored in both time and space(Figure5-28).

D N A M i c r o a r r a y sC a nB e U s e dt o E v a l u a t et h e E x p r e s s i o on f M a n y G e n e sa t O n e T i m e Monitoring the expressionof thousandsof genessimultaneously is possiblewith DNA microarray analysis,another technique based on the concept of nucleic acid hybridization. A DNA microarray consists of an organized array of thousands of individual, closely packed gene-specific sequencesattached to the surfaceof a glassmicroscopeslide. By coupling microarray analysis with the results from genome sequencing projects, researchers can analyze the global patterns of gene expression of an organism during specificphysiological responsesor developmentalprocesses. Preparation of DNA Microarrays In one methodfor preparing microarrays,an =1-kb portion of the coding region of each gene analyzedis individually amplified by the PCR. A robotic deviceis used to apply each amplified DNA sample to the surface of a glass microscope slide, which then is chemically processedto permanently attach the DNA sequencesto the glasssurfaceand to denature them. A typical array might contain =6000 spotsof DNA in a2 x 2- cm grid. In an alternative merhod, multiple DNA oligonucleotides, usually at least 20 nucleotides in length, are synthesizedfrom an initial nucleotide that is covalently bound to the surface of a glass slide. The synthesisof an oligonucleotide of specific sequencecan be programmed in a small region on the surfaceof the slide. Severaloligonucleotidesequencesfrom a singlegeneare thus synthesizedin neighboring regions of the slide to analyzeexpressionof that gene. Vith this method, oligonucleotidesrepresentingthousands of genescan be produced on a singleglassslide. Becausethe methods for constructing these arrays of synthetic oligonucleotideswere adapted from methods for manufacturing microscopic integrated circuits used in computers, these types of oligonucleotidemicroarrays are often called DNA chips.

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A EXPERIMENTAL FIcURE5-27 Northernblot analysisreveals increased expression of p-globinmRNAin differentiated erythroleukemia cells.ThetotalmRNAin extracts of erythroleukemia cellsthatweregrowingbut uninduced andin cells induced to stopgrowingandallowedto differentiate for 4g hoursor 96 hourswasanalyzed by Northern blottingfor B-globin mRNAThe density of a bandisproportional to theamountof mRNApresent TheB-globin mRNAisbarelydetectable in uninduced cells(UNlane) but increases morethan1000-fold by 96 hoursafterdifferentiation is induced[Courtesy of L Kole] 192

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Using Microarrays to Compare Gene Expression under Different Conditions The initial stepin a microarrayexpression study is to preparefluorescentlylabeledcDNAs corresponding to the mRNAs expressedby the cellsunder study.When the cDNA preparation is applied to a microarray,sporsrepresenting genesthat are expressedwill hybridize under appropriate conditions to their complementarycDNAs in the labeledprobe mix, and can subsequentlybe detectedin a scanninglasermicroscope. Figure 5-29 depicts how this method can be applied to examine the changes in gene expression observed after starved human fibroblasts are transferred to a rich, serumcontaining, growth medium. In this type of experiment, the separatecDNA preparations from starvedand serum-grown

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A EXPERIMENTAL FIGURE 5-28 In situ hybridizationcandetect activityof specificgenesin whole and sectionedembryos.The s p e c i m ei n s p e r m e a b i l i zbeydt r e a t m e nwt i t h d e t e r g e natn da protease to expose the mRNAto the probeA DNAor RNAprobe, s p e c i f ifco r t h e m R N Ao f i n t e r e sits, m a d ew i t h n u c l e o t i daen a l o g s c o n t a i n i ncgh e m i c aglr o u p tsh a tc a nb e r e c o g n i z ebdy a n t i b o d i e s A f t e rt h e p e r m e a b i l i zsepde c i m ehna sb e e ni n c u b a t ewdi t ht h e p r o b eu n d e cr o n d i t i o nt h s a tp r o m o t e hybrid z a t i o nt,h ee x c e s s p r o b ei s r e m o v ew d i t ha s e r i eosf w a s h e sT h es p e c i m ei n st h e n i n c u b a t ei d n a s o l u t i ocno n t a i n i nagn a n t i b o dtyh a tb i n d st o t h e p r o b eT hs a n t i b o diysc o v a i e n tl loyi n e dt o a r e p o r t eern z y m (ee g , se h o r s e r asdh p e r o x i d a o r a l k a l i npeh o s p h a t a st hea) tp r o d u c eas productAfterexcess coloredreaction antibodyhasbeenremoved,

drecipitate o r t h e r e p o r t eern z y m iesa d d e dA c o l o r e p s u b s t r a tf e f o r m sw h e r et h e p r o b eh a sh y b r i d i z et od t h e m R N Ab e i n g d e t e c t e d( a )A w h o l em o u s e m b r y oa t a b o u t1 0 d a y so f probedfor Sonichedgehog mRNAThestainmarks development ( r e d a l o n gt h e m e s o d e r rmu n n i n g a r o d o f a r r o w ) , t h en o t o c h o r d f u t u r es p i n acl o r d ( b )A s e c t i oonf a m o u s e m b r y os i m i l atro t h a t i n o a r t( a ) T h ed o r s a l / v e n tar xailso f t h e n e u r atlu b e( N T c) a nb e (redarrow) notochord seen,with the Sonichedgehog-expressing ( b l u e v e n t r a (l c )A s t i l f l a r t h e r a r r o w ) e n d o d e r m b e l o wi t a n dt h e during embryoprobedfor an mRNAproduced wholeDrosophila is patternof bodysegments Therepeating tracheadevelopment of L visibleAnterior(head)is up,ventralisto the left Icourtesy andN/ P Scott Milenkovic l

fibroblastsare labeledwith differently colored fluorescent dyes.A DNA array comprising8600 mammaliangenesthen is incubatedwith a mixture containingequal amountsof the t w o c D N A p r e p a r a t i o n su n d e r h y b r i d i z a t i o nc o n d i t i o n s . After unhybridizedcDNA is washed away, the intensity of green and red fluorescenceat each DNA spot is measured microscopeand storedin computerfiles using a fluorescence under the name of eachgeneaccordingto its known position on the slide.The relativeintensitiesof red and greenfluorescencesignalsat eachspot are a ffreasureof the relativelevel of expressionof that genein resp()nseto serum.Genesthat are not transcribedunder thesegrowth conditions give no detectablesignal. Genesthat are transcribed at the same level under both conditions will hybridize equally to both r e d a n d g r e e n - l a b e l e dc D N A p r e p a r a t i o n s .M i c r o a r r a y analysisof geneexpressionin fibroblastsshowedthat transcriptionof about 500 of the 8600 genesexaminedchanged substantiallyafter addition of serum.

the many different changesin cell physiology that occur when cells are transferred from one medium to another. I n o t h e r w o r d s , g e n e st h a t a p p e a rt o b e c o - r e g u l a t e di n a s i n g l e m i c r o a r r a y e x p r e s s i o ne x p e r i m e n t m a y u n d e r g o changesin expressionfor very different reasonsand may a c t u a l l y h a v e v e r y d i f f e r e n t b i o l o g i c a l f u n c t i o n s .A s o l u t i o n t o t h i s p r o b l e m i s t o c o m b i n et h e i n f o r m a t i o n f r o m a set of expressionarray experimentsto find genesthat are similarly regulated under a variety of conditions or over a oeriod of time. This more informative use of multiple expressionarray experiments is illustrated by examining the relative expression of the 8600 genesat different times after serum addition, generatingmore than 104 individual piecesof data. A computer program, relatedto the one usedto determinethe can organizethese of differentprotein sequences' relatedness over the expression genes show similar that data and cluster cluster such Remarkably, serum addition. after time course particproteins genes whose encoded groups sets of analysis ipate in a common cellular process, such as cholesterol biosynthesisor the cell cycle (Figure5-30).

C l u s t e rA n a l y s i so f M u l t i p l e E x p r e s s i o n E x p e r i m e n t sl d e n t i f i e sC o - r e g u l a t e G d enes F i r m c o n c l u s i c l n sr a r e l y c a n b e d r a w n f r o m a s i n g l e m i c r o a r r a y e x p e r i m e n t a b o u t w h e t h e r g e n e st h a t e x h i b i t s i m i l a r c h a n g e si n e x p r e s s i o na r e c o - r e g u l a t e da n d h e n c e likely to be closely related functionally. For example, m a n y o f t h e o b s e r v e dd i f f e r e n c e si n g e n e e x p r e s s i o nj u s t e fs d e s c r i b e di n f i b r o b l a s t sc o u l d b e i n d i r e c t c o n s e < r u e n c o

analysiswill be a powerful F.i In the future, microarray di"enustic tool in medicine. For instance,particular fiil setsof .RXRt have beenfound to distinguishtumors with a poor prognosisfrom those with a good prognosis.Previously indistinguishablediseasevariations are now detectable' Analysisof tumor biopsiesfor thesedistinguishingmRNAs

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Technique Animation:Synthesizing an Oligonucleotide Array flllt TechniqueAnimation: Screeningfor Patternsof Gene Expression Fibroblasts w i t h o u ts e r u m

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< EXPERIMENTAL FIGURE 5-29 DNAmicroarrayanalysiscan revealdifferencesin gene expressionin fibroblastsunder differentexperimentalconditions.(a)In thisexample, cDNA prepared frommRNAisolated fromfibroblasts eitherstarved for serumor afterserumadditionislabeled with different fluorescent dyesA microarray composed of DNAspotsrepresenting 8600 genesisexposed mammalian to an equalmixture of thetwo cDNA preparations underhybridization conditions Theratioof the intensities of redandgreenfluorescence overeachspot,detected with a scanning confocal lasermicroscope, indicates the relative expression of eachgenein response to serum.(b)A micrograph of a smallsegment of an actualDNAmicroarray. Eachspotin this 16 x genehybridized 16arraycontains DNAfroma different to control andexperimental cDNAsamples labeled with redandgreen fluorescent dyes(A yellowspotindicates equalhybridization of greenandredfluorescence, indicating no changein gene (b)Alfred expression) Pasieka/Photo Researchers, Inc] [Part

Wash Measuregreen and red fluorescenceover eachspot

will help physiciansto selectthe most appropriate treatment. As more patternsof geneexpressioncharacteristicof various diseasedtissuesare recognized,the diagnostic use of DNA microarrays will be extendedto other conditions.I

E. coliExpressionSystemsCan ProduceLarge Quantitiesof Proteinsfrom ClonedGenes

A lf a spot is green,expressionof that gene decreasesin cells after serum addition

.E l f a s p o t i s r e d ,e x p r e s s i o no f t h a t g e n e i n c r e a s e isn c e l l s after,seru m,addition

Many protein hormones and other signalingor regulatory proteins are normally expressedat very low concentrations,precluding their isolation and purification in large quantities by standard biochemical techniques. Widespread therapeutic use of such proteins, as well as basicresearchon their structureand functions, dependson efficient proceduresfor producing them in large amounts at reasonablecost. RecombinantDNA techniquesthat turn E. coli cells into factories for synthesizinglow-abundance proteins now are used to commercially produce granulocyte colony-stimulating factor (G-CSF), insulin, growth hormone, and other human proteins with therapeuticuses. For example, G-CSF stimulatesthe production of granulocytes, the phagocytic white blood cells critical to defense against bacterial infections. Administration of G-CSF to cancer patients helps offset the reduction in granulocyte production caused by chemotherapeuticagents, thereby protecting patients againstseriousinfection while they are receiving chemotherapy.I The first step in producing large amounts of a lowabundanceprotein is to obtain a cDNA clone encoding the full-length protein by methods discussedpreviously.The second step is to engineerplasmid vectorsthat will expresslarge amounts of the encoded protein when it is inserted into E. coli cells.The key to designingsuch expressionvectors is inclusion of a promoter, a DNA sequencefrom which transcription of the cDNA can begin. Consider,for example, the

194

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A A EXPERIMENTAL FIGURE 5-30 Clusteranalysisof data from multiple microarrayexpressionexperimentscan identify cogeneswas of 8600mammalian regulatedgenes.Theexpression at timeintervals overa 24-hour detected by microarray analysis periodafterserum-starved wereprovided with serum.The fibroblasts that algorithm diagram shownhereisbasedon a computer cluster groupsgenesshowingsimilar with a compared changes in expression overtime.Eachcolumnof colored serum-starved controlsample a time boxesrepresents a singlegene,andeachrow represents point.A redboxindicates relative to the in expression an increase in expression; anda blackbox,no control;a greenbox,a decrease

relatively simple system for expressing G-CSF shown in Figure 5-31. In this case,G-CSF is expressedin E. coli transformed with plasmid vectors that contain the lac promoter adjacentto the cloned cDNA encoding G-CSF.Transcription from the lac promoter occurs at high rates only when lactose, or a lactose analog such as isopropylthiogalactoside (IPTG), is added to the culture medium. Even larger quantities of a desired protein can be produced in more complicated E. coli expressionsystems. To aid in purification of a eukaryotic protein produced in an E. coli expression system, researchersoften modify the cDNA encodingthe recombinant protein to facilitate its separation from endogenousE. coli proteins. A commonly used modification of this type is to add a short nucleotide sequenceto the end of the cDNA, so that the expressedprotein will have six histidine residuesat the C-terminus. Proteins modified in this way bind tightly to an affinity matrix > EXPERIMENTAL FIGURE 5-31 Someeukaryoticproteinscan be producedin E coli cellsfrom plasmidvectorscontainingthe vectorcontainsa fragment lac promoter.(a)Theplasmidexpression the /acpromoterandthe containing of the E colichromosome analogIPTG, of the lactose lacZgene.Inthe presence neighboring /acZ normallytranscribes the iacZgene,producing RNApolymerase protein, intotheencoded mRNA,whichistranslated B-galactosidase (b)The/acZgenecanbe cut out of the expression vectorwith andreplaced by a clonedcDNA,in thiscaseone restriction enzymes granulocyte Whenthe factor(G-CSF). colony-stimulating encoding plasmid intoE.collcells,additionof IPTG istransformed resulting produce G-CSF fromthe/acpromoter transcription andsubsequent protein intoG-CSF mRNA.whichistranslated

at thetop The"tree"diagram icantchangein expression. signif genescanbe patterns for individual showshowthe expression to grouptogetherthe geneswith fashion in a hierarchical organized overtime'Five of expression in theirpatterns similarity the greatest geneswereidentified in this regulated of coordinately clusters by the barsat the bottom.Eachcluster asindicated experiment, proteins functionin a geneswhoseencoded multiple contains (A),thecellcycle process: biosynthesis cholesterol particular cellular (C),signaling andangiogenesis (B),the immediate-early response (E).[Courtesy of Michael (D),andwoundhealing andtissueremodeling Laboratoryl National Berkeley Lawrence B Eisen,

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G E N EE X P R E S S I O N TO STUDY U S I N GC L O N E DD N A F R A G M E N T S

195

that contains chelated nickel atoms, whereas most E. coli proteins will not bind to such a matrix. The bound proteins can be releasedfrom the nickel atoms by decreasingthe pH of the surrounding medium. In most cases,this procedure yields a pure recombinant protein that is functional, since addition of short amino acid sequencesto either the Cterminusor the N-terminus of a protein usually doesnot interfere with the protein's biochemicalactivity.

{a) Transient transfection cDNA

I transtectcultured I c e l l sb y l i p i dt r e a t m e n t or electroporation J

PlasmidExpressionVectorsCan Be Designedfor U s ei n A n i m a lC e l l s \Vhile bacterial expressionsystemscan be used successfully to createlarge quantitiesof someproteins, bacteriacannot be used in all cases.Many experimenrsto examine the function of a protein in an appropriate cellular context requlre expression of a geneticallymodified protein in cultured animal cells. Genesare cloned into specializedeukaryotic expressionvec, tors and are introduced into cultured animal cells by a processcalled transfection.Two common methods for transfecting animal cells differ in whether the recombinant vecor DNA is or is not integratedinto the host-cellgenomic DNA. In both methods,culturedanimal cellsmust be treatedto facilitatetheir initial uptake of the recombinantplasmidvector. This can be done by exposingcells to a preparationof lipids that penetrarethe plasma membrane, increasingits permeability to DNA. Alternatively, subjecting cells to a brief electric shock of severalthousand volts, a technique known as electroporation, makes them transiently permeableto DNA. Usuallythe plasmidDNA is addedin sufficient concentrationto ensurethat a large proportion of the cultured cellswill receiveat leastone copy of the plasmidDNA. Researchers have also harnessedvirusesfor their use in the laboratory; virusescan be modified ro contain DNA of interest, which is then introduced into host cells by simply infecting them with the recombinant virus. Transient Transfection The simplestof the two expression methods, calledtransient transfectioz, employs a vector similar to the yeast shuttle vectors describedpreviously. For use in mammalian cells,plasmid vectorsare engineeredalso to carry an origin of replicationderivedfrom a virus that infecrsmammalian cells, a strong promoter recognized by mammalian RNA polymerase,and the cloned cDNA encodingthe protein to be expressedadjacentto the promoter (Figure5-32a). Once such a plasmid vector entersa mammalian cell, the viral origin of replication allows it to replicate efficientl5 generating numerousplasmidsfrom which the protein is expressed.However,duringcell divisionsuchplasmidsare nor faithfullysegregatedinto both daughtercellsand in time a substantialfraction of the cells in a culture will not contain a plasmid, hencethe name t ransi ent t ransfecti on. Stable Transfection (Transformation) If an introduced vector integratesinto the genomeof the host cell, the genomeis permanentlyalteredand the cell is said to be transformed.lntegration most likely is accomplishedby mammalian enzymes that normally function in DNA repair and recombination. 196

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Proteinis expressedfrom cDNA integrated i n t o h o s tc h r o m o s o m e A EXPERIMENTAL FIGURE5-32 Transientand stable transfection with specially designed plasmid vectors permit expression of cloned genes in cultured animal cells.Bothmethodsemployplasmid vectorsthat containthe usualelements-ORl,selectablemarker(e.g , amp'), and polylinker-that permitpropagationin E. coli and inserlionof a clonedcDNAwith an adjacentanimalpromoter.Forsimplicity, these elementsarenot depicted(a)In transient transfection, the plasmid vectorcontainsan originof replication for a virusthat can replicatern the culturedanimalcells Sincethe vectoris not incorporated into the genome of the culturedcells,production of the cDNA-encoded proteincontinues onlyfor a limitedtime (b) In stabletransfection, the vectorcarries a selectable markersuchas neo',whichconfersresistance to G-418,The relatively few transfectedanimalcellsthat integratethe exogenousDNA into theirgenomesareselected on mediumcontainingG-418 Because the vectoris integratedinto the genome,thesestablytransfected,or transformed, cellswill continueto producethe cDNA-encoded protein as longasthe cultureis maintainedSeethe textfor discussion

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AUTOSOMAL RECESSIVE Sickle-cellanemia

Abnormal hemoelobin causesdeformation of red blood cells, which can become lodged in capillaries; also confers resistanceto malaria'

11625of sub-SaharanAfrican origin

Cysric fibrosis

Defective chloride channel (CFTR) in epithelial cells leads to excessivemucus in lungs.

1'12500of European origin

Phenylketonuria (PKU)

Defective enzyme in phenylalanine metabolism (tyrosine hydroxylase) results in excessphenylalanine, leading to mental retardation, unless restricted by diet.

1/10,000 of European origin

Tay-Sachsdisease

Defective hexosaminidaseenzyme leads to accumulation of excesssphingolipids in the lysosomesof neurons, impairing neural development.

1/1000 easternEuropean Jews

Huntington's disease

Defective neural protein (huntingtin) may assembleinto aggregatescausing damage to neural ttssue.

1/10,000of Europeanorigin

Hypercholesterolemia

Defective LDL receptor leads to excessivecholesterol in blood and early heart attacks.

tlI22French Canadians

Duchenne muscular dystrophy (DMD)

Defective cytoskeletal protein dystrophin leads to impaired muscle function.

1/3500 males

Hemophilia A

Defective blood clotting factor VIII leads to uncontrolled bleedine.

1-2110,000 males

AUTOSOMAL DOMINANT

X-LINKED RECESSIVE

provide clues to the molecular and cellular causeof the disease.Historically, researchershave usedwhatever phenotypic clues might be relevant to make guessesabout the molecular basis of inherited diseases.An early example of successful guessworkwas the hypothesisthat sickle-cellanemia,known to be a diseaseof blood cells,might be causedby defectivehemoglobin. This idea led to identification of a specificamino acid substitution in hemoglobin that causespolymerization of the defectivehemoglobin molecules,causingthe sickle-like deformation of red blood cellsin individuals who have inherited two copiesof the Hb' allele for sickle-cellhemoglobin. Most often, however,the genesresponsiblefor inherited diseasesmust be found without any prior knowledge or reasonablehypothesesabout the nature of the affectedgeneor its encodedprotein. In this section,we will seehow human geneticistscan find the generesponsiblefor an inherited disease by following the segregationof the diseasein families' The segregationof the diseasecan be correlatedwith the segregation of many other genetic markers, eventually leading to identification of the chromosomalposition of the affected

gene.This information, along with knowledgeof the sequence of th. hn-".t genome,can ultimately allow the affectedgene mutations to be pinpointed. and the disease-causing

s howOneof Three M a n y I n h e r i t e dD i s e a s e S Major Patternsof Inheritance Human genetic diseasesthat result from mutation in one specific gene exhibit severalinheritance patterns depending on the nature and chromosomal location of the alleles that causethem. One characteristicpattern is that exhibited by a dominant allele in an autosome (that is, one of the 22 human chromosomes that is not a sex chromosome). Becausean autosomal dominant allele is expressed in the heterozygote,usually at least one of the parents of an affected individual will also have the disease'It is often the case that the diseasescaused by dominant alleles appear later in life after the reproductive age. If this were n o i t h e c a s e , n a t u r a l s e l e c t i o nw o u l d h a v e e l i m i n a t e d the allele during human evolution. An example of an EENES H U M A N D I S E A SG I D E N T I F Y I NAGN D L O C A T I N G

.

199

(al Autosomal dominant: Huntington's disease

d

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FIGURE 5-35 Threecommoninheritancepatternsfor human geneticdiseases. Wild-type (A)andsexchromosomes autosomal (X andY)areindicated by superscript plussigns(a)In an autosomal d o m i n a ndti s o r d es ru c ha sH u n t i n g t osnd i s e a soen, l yo n em u t a n t alleleisneeded to conferthe diseaself eitherparentis heterozygous for the mutantHDallele,hisor herchildren havea 50 percent c h a n coef i n h e r i t i nt h g em u t a nat l l e l e a n dg e t t i n g t h ed i s e a s(eb )l n an autosomal recessive disorder suchascystic fibrosis, two mutant alleles mustbe present to conferthe diseaseBothparents mustbe heterozygous carriers of the mutantCFIRgenefor theirchildren to be at riskof beingaffected or beingcarriers(c)An X-linked recessive disease suchasDuchenne muscular dystrophy iscauseo oy a recessive mutation on theX chromosome andexhibits thetypicalsex_ linkedsegregatron patternMalesbornto mothers heterozygous for a mutantDMDallelehavea 50 percent chance of inheriting the mutantalleleandbeingaffectedFemales bornto heterozvqous mothers havea 50 percent chance of beinqcarriers

a u t o s o m a l d o m i n a n t d i s e a s ei s H u n t i n g t o n ' s d i s e a s e ,a neural degenerativediseasethat generally strikes in midto late life. If either parenr carries a murant HD allele, e a c h o f h i s o r h e r c h i l d r e n ( r e g a r d l e s so f s e x ) h a s a 5 0 percent chance of inheriting rhe mutanr allele and being a f f e c t e d( F i g u r e5 - 3 5 a ) . A recessiveallelein an autosomeexhibits a quite different segregatronpattern. For an autosomal recessiueallele, both parentsmust be hererozygouscarriersof the allelein order for their children to be at risk of being affectedwith the disease. Each child of heterozygousparenrshas a 25 percentchanceof receiving both recessiveallelesand thus being affected,a 50 percentchanceof receivingone normal and one mutant allele and thus being a carrier, and a 25 percentchanceof receiving two normal alleles.A clear exampleof an autosomalrecessive diseaseis cystic fibrosis, which results from a defectivechloride-channelgeneknown as CFTR (Figure5-35b). Relatedindividuals (e.g.,first or secondcousins)have a relatively high 200

.

c H A p r EsR I

probability of being carriers for the same recessivealleles. Thus children born to related parents are much more likely than those born to unrelated parents to be homozygousfor, and thereforeaffectedby, an autosomalrecessivedisorder. The third common pattern of inheritanceis that of an XLinked recessiueallele. A recessiveallele on the X chromosome will most often be expressedin males,who receiveonly one X chromosome from their mother, but not in females, who receivean X chromosome from both their mother and their father. This leadsto a distinctive sex-linked segregation pattern where the diseaseis exhibited much more frequently in males than in females.For example, Duchenne muscular dystrophy(DMD), a muscledegenerative diseasethat specifically affects males, is causedby a recessiveallele on the X chromosome. DMD exhibits the typical sex-linked segregation pattern in which mothers who are heterozygous and therefore phenotypically normal can act as carriers, rransmitting the DMD allele, and therefore the disease,to 50 percent of their male progeny (Figure5-35c).

M o L E c u L AGRE N E Tr tEcc H N l o u E s

D N A P o l y m o r p h i s mAs r e U s e di n L i n k a g e M a p p i n gH u m a nM u t a t i o n s Once the mode of inheritancehas beendetermined,the next step in determining the position of a diseaseallele is to genetically map its position with respect to known genetic markers using the basic principle of genetic linkage as described in Section5.1. The presenceof many different already mapped genetic rraits, or markers, distributed along the length of a chromosome facilitatesthe mapping of a new mutation by assessingits possiblelinkage to these marker genesln appropriate crosses.The more markers that are available,the more preciselya mutarion can be mapped.The density of genetic markers neededfor a high-resolution human genetic map is about one marker every 5 centimorgans (cM) (as discussedpreviously,one geneticmap unir, or centimorgan, is defined as the distance between two positions along a chromosome that results in one recombinant individual in 100 progeny).Thus a high-resolutiongenericmap requires25 or so geneticmarkersof known position spread along the length of each human chromosome. In the experimentalorganismscommonly used in genetic studies,numerous markers with easily detectablephenotypes are readily availablefor geneticmapping of mutations.This is not the casefor mapping geneswhose mutant allelesare associated with inherited diseasesin humans. However" recombinant DNA technologyhas made availablea wealth of useful DNA-based molecular markers. Becausemost of the human genomedoesnot code for protein, alarge amount of sequence variation exists betweenindividuals. Indeed, it has been estimated that nucleotidedifferencesbetweenunrelatedindividuals can be detectedon an averageof every 103 nucleotides.If thesevariations in DNA sequence,referredto as DNA polymorphisms,can be followed from one generationto the next, they can serve as genetic markers for linkage studies. Currently, a panel of as many as 104 different known polymorphisms whose locations have been mapped in the human genomeis usedfor geneticlinkage studiesin humans.

Restriction fragment length polymorphisms (RFLPs) were the first type of molecular markers used in linkage studies.RFLPsarisebecausemutationscan createor destroy the sitesrecognizedby specificrestriction enzymesthat happen to lie in human DNA, leadingto variationsbetweenindividuals in the length of restriction fragments produced from identical regionsof the genome.Differencesin the sizes of restriction fragments betweenindividuals can be detected by Southern blotting with a probe specific for a region of DNA known to contain an RFLP (Figure5-36a).The segregation and meiotic recombination of such DNA polymorphisms can be followed like typical genetic markers. Figure 5-36b illustrates how RFLP analysis of a family can detect the segregaticlnof an RFLP that can be used to test for statistically significant linkage to the allele for an inherited diseaseor some other human trait of interest. The amassedgenomic sequenceinformation from different humans has led to identification of other useful DNA polymorphisms in recent years. Single-nwcleotidepolymorphisms (SNPs) constitute the most abundant type and are therefore useful for constructing geneticmaps of maximum resolution. Another useful type of DNA polymorphism consistsof a variable number of repetitionsof a one- two-, or Suchpolymorphisms,known as simple three-basesequence. sequencerepeats (SSRs)or microsatellites, presumably are formed by recombinationor a slippagemechanismof either the template or newly synthesizedstrands during DNA Hybridization banding pattern from individual with both allele 1 and allele2

lat

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replication. A useful property of SSRsis that different individuals will often have different numbers of repeats.The existenceof multiple versionsof an SSRmakes it more likely to produce an informative segregationpattern in a given pedigreeand therefore be of more generalusein mapping the positionsof diseasegenes.If an SNP or SSRaltersa restriction site, it can be detectedby RFLP analysis. More commonly' however, these polymorphisms do not alter restriction fragments and must be detectedby PCR amplification and DNA sequenclng.

Geneswith a LinkageStudiesCanMap Disease Resolutionof About 1 Centimorgan lVithout going into all the technical considerations,let's see how the allele conferring a particular dominant trait (e.9., familial hypercholesterolemia)might be mapped. The first step is to obtain DNA samplesfrom all the members of a family containing individuals that exhibit the disease.The DNA from each affected and unaffected individual then is analyzed to determine the identity of a large number of known DNA polymorphisms (either SSR or SNP markers can be used). The segregationpattern of each DNA polymorphism within the family is then compared with the segrigation of the diseaseunder study to find those polymorphisms that tend to segregatealong with the disease' Finaily, computer analysisof the segregationdata is used to (b) Grandparents

G randparents

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FIGURE5-36 Restrictionfragment length A EXPERIMENTAL can be followed like genetic markers. polymorphisms (RFLPs) (a) In the two homologouschromosomes shown,DNA is treatedwith enzymes(A and B),which cut DNA at two differentrestriction (a and b) The resultingf ragmentsare subjected differentsequences probe (seeFigure5-26)with a radioactive to Southernblot analysis that bindsto the indicatedDNA region(green)to detectthe betweenthe two homologous fragments Sinceno differences by the B enzyme, recoqnized occurin the sequences chromosomes by the probe,as indicatedby a only one fragmentis recognized band However,treatmentwith enzymeA singlehybridization producesradiographically distinctf ragmentsof two differentlengths

tr

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(a1-a2anda1-a3),and two bandsare seen,indicatingthat a mutationhascausedthe lossof one of the a sitesin one of the two of the DNA from a analysis chromosomes(b) Pedigreebasedon RFLP regionknown to be presenton chromosome5. The DNA samples enzymeIaql and analyzedby Southern were cut with the restriction of the genomeexistsin threeallelic region this family, blotting In this by Iaql sitesspaced10, 7 '7, or 6 5 kb apart forms characterized Eachindividualhastwo alleles;somecontainallele2 (7 7 kb) on both at this site Circles and othersare heterozygous chromosomes, gel lanesare in the The males indicate indicatefemales;squares and are generally pedigree above in the the subjects as order same , e l5l 1 : 3 1 ]9 l l tearl , 1 9 8 7C r D o n i s - K ee a l i g n e db e l o wt h e m l A f t e H . I D E N T I F Y IA NN GD L O C A T I NHGU M A ND I S E A SGEE N E S

201

calculatethe likelihood of linkage between each DNA polymorphism and the disease-causing allele. In practice, segregation data are collected from different families exhibiting the same diseaseand pooled. The more families exhibiting a parricular diseasethaican be examined, the greater the statistical significance of evidence for linkage that can be obtained and the grearerthe precisionwith which the distancecan be measuredbetween a linked DNA oolvmorphism and a diseaseallele.Most family studieshave a maximum of about 100 individualsin which linkage betweena diseasegene and a panel of DNA polymorphismscan be tested. This number of individuals setsthe practical upper limit on the resolutionof sucha mapping study to about 1 cenrimorgan,or a physicaldistanceof about 7.5 x 10s basepairs. A phenomenoncalledlinkagedisequilibriumisthe basisfor an alternative strategy,which in some casescan afford a higher degreeof resolution in mapping studies.This approach dependson the particular circumstancein which a genericdisease commonly found in a particular population resultsfrom a single mutation that occurredmany generationsin the past. The DNA polymorphismscarriedby this ancestralchromosomeare collectively known as the haplotype of that chromosome. As the diseasealleleis passedfrom one generationto the next, only the polymorphisms that are closestto the diseasegenewill not be separatedfrom it by recombination. After many generarlons the region that contains the diseasegenewill be evident because this will be the only region of the chromosome thar will carry the haplotype of the ancestralchromosome conservedthrough many generations(Figure5-37).By assessing the distributionof specificmarkersin all the affectedindividualsin a population, geneticistscan identify DNA markers tightly associatedwith

appearedon the ancestralchromosome-in somecasesthis can amount to finding markersthat are so closelylinked to the diseasegenethat even after hundredsof meiosesthey have never beenseparatedby recombinarion.

FurtherAnalysisls Neededto Locatea Disease G e n ei n C l o n e dD N A Although linkage mapping can usually locare a human diseasegene to a region containing about 105 base pairs, as many as 10 different genesmay be located in a region of this size. The ultimate objective of a mapping study is to locate the genewithin a cloned segmenrof DNA and then to determine the nucleotide sequenceof this fragment. The relative scalesof a chromosomalgenetic-ap anJphysicalmaps corresponding to ordered sers of plasmid clones and the nucleotidesequenceare shown in Figure5-38. One strategyfor further localizinga diseasegenewithin the genome is to identify mRNA encoded by DNA in the region of the geneunder study. Comparison of geneexpressionin tissues 2O2

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A FIGURE 5-37 Linkagedisequilibrium studiesof human populationscan be usedto map genesat high resolution.A newdisease mutation willarisein the contextof an ancestral chromosome amonga setof polymorphisms known asthehaplotype (indicated by pinkshading). Aftermanygenerations, chromosomes thatcarrythe disease mutation willalsocarrysegments of the ancestral haplotype thathavenot beenseparated fromthe disease mutationby recombination Thebluesegments of these chromosomes general represent haplotypes derived fromthegeneral population andnotfromtheancestral haplotype in whichthe mutatron originally aroseThisphenomenon is knownaslinkage disequilibrium Thepositionof the disease mutationcanbe located by scanning chromosomes containing thedisease mutation for highly polymorphisms conserved corresponding to theancestral haplotype from normal and affected individuals may suggesttissuesin which a particular diseasegenenormally is expressed.For instance,a mutation that phenotypically affects muscle, but no other tissue,might be in a genethat is expressedonly in muscle tissue. The expressionof mRNA in both normal and affected individuals generally is determined by Northern blotting or in situ hybridization of labeledDNA or RNA to tissuesections. Northern blots, in situ hybridization, or microarray experiments permit comparison of both the level of expression and the size of mRNAs in mutant and wild-type tissues.Although the sensitivity of in situ hybridization is lower than that of Northern blot analysis,it can be very helpful in identifying an mRNA that is expressedat low levels in a given tissue but at very high levels in a subclassof cells within that tissue.An mRNA that is altered or missing in various individuals affected with a diseasecomparedwith wild-type individualswould be an excellent candidate for encoding the protein whose disrupted function causesthat disease. In many cases,point mutations that give rise to diseasecausing allelesmay result in no detectablechange in the level of expression or electrophoretic mobility of mRNAs. Thus if comparison of the mRNAs expressedin normal and affected individuals reveals no detectabledifferencesin the candidate mRNAs, a search for point mutarions in the

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M E T H O DO F Linkageto restriction DETECTION:Chromosome b a n d i n gp a t t e r n f r a g m e n tl e n g t hp o l y morPhismsRFLPS, in Fluorescence s i t u h y b r i d i z a t i o n s i n g l en u c l e o t i d ep o l y (FISH) m o r P h i s m sS N P s ,a n d s l m p l es e q u e n c e repeatsSSRs

5-38 The relationshipbetweenthe geneticand A FIGURE a deptcts Thediagram physicalmapsof a humanchromosome' of detailThe levels at different analyzed humanchromosome when asa wholecanbeviewedin the lightmicroscope chromosome andthe at metaphase, statethatoccurs it isin a condensed by canbe determined ic sequences locationof specif approximate (FISH) At the nextlevelof detail, in sltuhybridization fluorescence

DNA regions encoding the mRNAs is undertaken. Now that highly efficientmethodsfor sequencingDNA are availfrequently determinethe sequenceof canable, researchers DNA isolatedfrom affectedindividualsto of regions didate identify point mutations. The overall strategyis to search for a coding sequencethat consistentlyshowspossiblydelereriousalterationsin DNA from individualsthat exhibit the disease.A limitation of this approachis that the region near the affectedgene may carry naturally occurring polymorphisms unrelated to the gene of interest. Such polymorphisms,not functionally related to the disease,can lead to misidentificationof the DNA fragmentcarrying the geneof interest.For this reason,the more mutant allelesavailable for analysis,the more likely that a gene will be correctly identified.

Sequence map

Hybridization Sanger (dideoxY) to plasmid sequenclng clones

2d to Genomes' FromGenes et al, 2003,Genetics: fromL Hartwell lAdapted Hill ed, McGraw I

s e s u l tf r o m M u l t i p l e M a n y I n h e r i t e dD i s e a s e R GeneticDefects Most of the inherited human diseasesthat are now understood at the molecular level are monogenetic traits; that is, a clearly discerniblediseasestate is produced by a defect in a single gene.Monogenic diseasescausedby mutation in one specific gene exhibit one of the characteristic inheritance patterns shown in Figure 5-35' The genes associatedwith most of the common monogenic diseases have already been mapped using DNA-based markers as describedpreviouslY. However, many other inherited diseasesshow more complicatedpatterns of inheritance,making the identification of the underlying genetic causemuch more difficult' EENES H U M A N D I S E A SG I D E N T I F Y I NAGN D L O C A T I N G

O

203

One type of added complexity that is frequently encountered is genetic heterogeneity.In such cases,mutations in any one of multiple different genes can cause the same disease.For example, retinitis pigmentosa,which is characterizedby degenerationof the retina usually leading to blindness,can be causedby mutations in any one of more than 60 different genes. In human linkage studies, data from multiple families usually must be combined to deter_ mine whether a statisricallysignificant linkage exists be_ t w e e n a d i s e a s eg e n e a n d k n o w n m o l e c u l a r m a r k e r s . Genetic heterogeneitysuch as rhar exhibited by retinitis pigmentosa can confound such an approach becauseany statisticaltrend in the mapping data from one family tends to be canceledout by the data obtained from another fam_ i l y w i t h a n u n r e l a t e dc a u s a t i v eg e n e . Human geneticisrsused two different approachesto _ identify the many genesassociatedwith retinitii pigmenrosa. The first approach relied on mapping studies in &ception_ ally large single families rhar contained a sufficient number of affectedindividuals to provide statisticallysignificant evidencefor linkage betweenknown DNA polymolphisms and a single causativegene. The genesidentified in such studies

retinitis pigmentosa.This approach of using additional in_ formation to focus screeningefforts on a s.rbiet of candidate genesled to identificationof additional rare causativemura_

ldentifying and Locating Human DiseaseGenes r Inherited diseasesand other traits in humans show t h r e e m a j o r p a t t e r n s o f i n h e r i t a n c e :a u t o s o m a l d o m i nant, autosomal recessive,and X-linked recessive(see F i g u r e5 - 3 5 ) . r Genesfor human diseasesand other trairs can be mapped by determining their cosegregation during meiosis with markers whose locations in the genome are known. The closer a geneis to a particular marker, the more likelv thev are to cosegregate, of human geneswith great precision requires of molecular markers distributed along the es. The most useful markers are differencesin the DNA sequence(polymorphisms) between individuals in noncoding regions of the genome. r DNA polymorphisms useful in mapping human genesinclude restriction fragment length polymorphisms (RFLps), single-nucleotidepolymorphisms (SNps), and simple sequencerepeats(SSRs). inkage mapping often can locate a human diseasegene a chromosomal region that includes as many as l0 genes.To identify the geneof interestwithin this candidate region_typicallyrequires expressionanalysis and compari_ son of DNA sequencesbetween wild-type and disiaseaffectedindividuals. r Some inherited diseasescan result from mutations in dif_ ferent genesin different individuals (geneticheterogeneity). The occurrence and severity of other diseasesdepend on the presenceof mutant allelesof multiple genesin the same individuals (polygenictraits). Mapping of the genesassociated with such diseasesis particularly difficult becausethe occurrenceof the diseasecannot readily be correlated to a singlechromosomallocus.

ff,l Inactivatingthe Functionof SpecificGenesin Eukaryotes The elucidation of DNA and protein sequencesrn recent y e a r s h a s l e d t o i d e n t i f i c a t i o no f m a n y g e n e s .u s i n g s e quencepatternsin genomicDNA and the sequencesimilar_ ity of the encodedproteins with proteins oi known function. As discussedin Chapter 6, the general functions of proteins identified by sequencesearchesmay be predicted by analogy with known proteins. However, the precisein vivo roles of such "new" proteins may be lrnclearin the ab_ senceof mutant forms of the correspondinggenes.In this

CHAPTER 5

J

MOLECULAG RENETIC TECHNIQUES

Three basic approachesunderlie these gene-inactivation techniques:(1) replacinga normal genewith other sequences, (2) introducing an allelewhose encodedprotein inhibits functioning of the expressednormal protein, and (3) promoting destructionof the mRNA expressedfrom a gene.The normal endogenousgeneis modified in techniquesbasedon the first approach but is not modified in the other approaches'

(a)

20-nt flanking sequence

.-f-hi"_l

s y n r h sr i s DNJA

-

NormalYeastGenesCan Be Replacedwith M u t a n t A l l e l e sb y H o m o l o g o u sR e c o m b i n a t i o n Modifying the genomeof the yeastS. cereuisiaeis particularly easy for two reasons:yeast cells readily take up exogenous DNA under certain conditions, and the introduced DNA is efficiently exchangedfor the homologous chromosomal site in the recipient cell. This specific,targeted recombination of identical stretchesof DNA allows any genein yeastchromosomesto be replacedwith a mutant allele. (As we discussin Section5.1, recombination between homologous chromosomesalso occurs naturally during meiosis.) In one popular method for disrupting yeast genesin this fashion, the PCR is used to generatea disruption construct containing a selectablemarker that subsequentlyis transfectedinto yeastcells.As shown in Figure 5-39a,primersfor PCR amplification of the selectablemarker are designedto flankinclude about 20 nucleotidesidenticalwith sequences amplified resulting The replaced. ing the yeast gene to be constructcomprisesthe selectablemarker (e.g.,the kdnMX gene,which llke neo' confers resistanceto G-41 8 ) flanked by about 20 basepairs that match the ends of the target yeast gene. Transformeddiploid yeast cells in which one of the two copiesof the target endogenousgenehas beenreplaced by the disruption construct are identified by their resistance phenotype.Theseheterozygous to G-418 or other selectable diploid yeastcellsgenerallygrow normally regardlessof the function of the target gene,but half the haploid sporesderived from thesecellswill carry only the disruptedallele(Figure 5-39b). If a geneis essentialfor viability,then sporescarrying a disruptedallelewill not survive. Disruption of yeastgenesby this method is proving parthe role of proteinsidentifiedby ticularly usefulin assessing analysisof the entire genomic DNA sequence(seeChapter 6). A large consortiumof scientistshas replacedeach of the approximately 6000 genesidentified by this analysiswith the kanMX disruption construct and determinedwhich gene disruptionslead to nonviablehaploid spores.Theseanalyses have shown that about 4500 of the 6000 yeastgenesare not

gene may be viable becauseof operation of backup or compensatorypathways.To investigatethis possibility,yeast geneticistscurrently are searchingfor syntheticlethal mutations that might reveal nonessentialgeneswith redundant functions(seeFigure 5-9c).

*Fri-"|.

-

Z

Disruption construct

(b)

Four haploid spores

FLI G U R E5 - 3 9 H o m o l o g o u s r e c o m b i n a t i o n a EXPERIMENTA constructs can inactivate specific disruption with transfected ( a ) y e a s t . A s u i t a b l ec o n s t r u cfto r d i s r u p t i n ga g e n e s i n target . h et w o p r i m e r s t a r g e tg e n ec a n b e p r e p a r e db y t h e P C R T d e s i g n e df o r t h i s p u r p o s ee a c hc o n t a i na s e q u e n c eo f a b o u t 2 0 yeast n u c l e o t i d e(sn t )t h a t i s h o m o l o g o u st o o n e e n d o f t h e t a r g e t o f D NA a s e g m e n t a m p l i f y t o n e e d e d s e q u e n c e s a s g e n ea s w e l l carryinga selectablemarkergene such as kanMX, which confers to G-418. (b) When recipientdiploid Saccharomyces resistance c e l l sa r et r a n s f o r m e dw i t h t h e g e n e d i s r u p t i o nc o n s t r u c t , h o m o l o g o u sr e c o m b i n a t i obne t w e e nt h e e n d so f t h e c o n s t r u c t s i l l i n t e g r a t et h e a n d t h e c o r r e s p o n d i ncgh r o m o s o m asl e q u e n c ew

nonvrable

O F S P E C I F IGCE N E SI N E U K A R Y O T E S I N A C T I V A T I NTGH E F U N C T I O N

205

(a) Formation of ES cells carrying a knockout mutation neor

fuHSV

G e n eX r e p l a c e m e nct o n s t r u c t

Homologous ,/ r. recombination,? "il" ./

r'

ES-ceilDNA

;

G e n eX

O t h e rg e n e s

I I Gene-targeted linsertion

I II R a n d o m ltnserilon

v

v

M u t a t i o ni n g e n eX

N o m u t a t i o ni n g e n eX

Cellsare resistantto G - 4 1 8a n d g a n c i c l o v i r

C e l l sa r e r e s i s t a ntto G - 4 1 8b u t s e n s i t i v e to ganciclovir

(b) Positive and negative selection of recombinant ES cells O OO 6+

N o n r e c o m b i n a ncte l l s Recombinantswith gene-targetedinsertion

| - r " . , w i t hG - 4 1 8 ( p o s i t i vsee l e c t i o n ) J

o.',oo o-oo Ag

I fr"u, with ganciclovir ( n e g a t i v es e l e c t i o n ) |

*

o, -o rro

< EXPERIMENTAL FIGURE 5-40 lsolationof mouseEScellswith a gene-targeteddisruption is the first stage in productionof knockoutmice.(a)Whenexogenous DNAisintroduced rnto embryonic stem(ES) cells,randominsertion vianonhomologous recombrnation occursmuchmorefrequently thangene-targeted insertion viahomologous recombination Recombinant cellsin which onealleleof geneX (orange andwhite)isdisrupted canbe obtained by usinga recombinant vectorthatcarries geneX disrupted with neo'(green), whichconfers resistance to G-418,and,outside the regionof homology, tkHsv(yellow), thethymidine genefrom kinase herpes simplex vrrusTheviralthymidlne kinase, unlikethe endogenous mouseenzyme, canconvert the nucleotide analog ganciclovir intothe monophosphate form;thisisthenmodified to t h et r i p h o s p h af o t er m ,w h i c hi n h i b i tcse l l u l aDrN Ar e p l i c a t i o i nnE S cellsThusganciclovir iscytotoxic for recombinant EScellscarrying the tkHsv gene.Nonhomologous insertion includes the tkHsv gene, whereas homologous insertion doesnot;therefore, onlycellswith nonhomologous insertion aresensitive to ganciclovir (b) Recombinant cellsareselected by treatment with G-41g,since cellsthatfailto pickup DNAor integrate it intotheirgenomeare sensitive to thiscytotoxic compoundThesurvivrng recombinant cells aretreatedwith ganciclovir Onlycellswith a targeted disruption in geneX, andtherefore lacklng geneanditsaccompanying the fkHsv cytotoxicity, willsurvive[See S L Mansour etal, 1988,Nature336.348l

A useful promoter for this purpose is the yeast GAL1 promoter, which is active in cells grown on galactose but completely inactive in cells grown on glucose. In this approach, the coding sequenceof an essentialgene (X) ligated to the GALl promoter is inserted into a yeast shuttle u..to. (seeFigure 5-17a).The recombinant vector then is introduced into haploid yeastcellsin which geneX has beendisrupted. Haploid cells that are transformed will grow on galactosemedium, since the normal copy of gene X on the vector is expressedin the presenceof galactose.\fhen the cells are transferredto a glucose-containingmedium, geneX no longer is transcribed;as the cells divide, the amount of

c'l

o o-

E S c e l l sw i t h t a r g e t e dd i s r u p t i o ni n g e n eX

Transcriptionof GenesLigatedto a Regulated P r o m o t e rC a n B e C o n t r o l l e dE x p e r i m e n t a l l y Although disruption of an essenrialgene required for cell g r o w t h w i l l y i e l dn o n v i a h l es p o r e sr.h i i m e r h o dp r o v i d e sl i m l e infclrmation about what the encodedprotein actually does in cells.To learn more about how a specificgenecontributesto cell growth and viability investigarorsmust be able to selec_ tively inactivatethe genein a population of growing cells.One method for doing rhis empkrysa regularedpro-oi.. to selec_ tively shut off transcription of an essentialgene. 206

o

c H A p r E5R I

M o L E c u L AGRE N E Tr tEcc H N t o u E S

In an early application of this method, researchersex, plored the function of cytosolic Hsc70 genes ln yeasr. Haploid cells with a disruption in all four redundant Hsc70 geneswere nonviable, unlessthe cells carried a vector containing a copy of the Hsc70 gene that could be ex_ pressedfrom the GALI promoter on galactosemedium. On transfer to glucose,the vector-carryingcells eventually stopped growing becauseof insufficient Hsc70 acivity. C a r e f u l e x a m i n a r i o n o f t h e s e d y i n g c e l l s r e v e a l e dt h a t their secretory proteins could no longer enter the endoplasmic rericulum (ER). This study provided the first evi_ dencefor the unexpectedrole of Hsc70 protein in translocation of secretory proteins into the ER, a process e x a m i n e di n d e t a i l i n C h a p t e r 1 3 .

#

uia"o: Microinjectionof ESCellsinto a Blastocyst

for a FIGURE 5-41 EScellsheterozygous > EXPERIMENTAL disruptedgene are usedto producegene-targetedknockout for a knockout cellsheterozygous stem(ES) mice.Step1: Embryonic (X andhomozygous for a dominant mutationin a geneof interest alleleof a markergene(here,browncoatcolor,A) aretransplanted thatarehomozygous cavityof 4 5-dayembryos intothe blastocoel alleleof the marker(here,blackcoatcolor,a) Step2: for a recessive female intoa pseudopregnant thenareimplanted Theearlyembryos by indicated cellsarechimeras, ES-derived containing Thoseprogeny micethenare theirmixedblackandbrowncoats.Step3: Chimeric fromthismatinghaveE5to blackmice;brownprogeny backcrossed of DNAisolated 4-6: Analysis derived cellsin theirgermline Steps brownmice canidentify froma smallamountof tailtissue of thesemice alleleIntercrossing for the knockout heterozygous that allele, produces for the disrupted homozygous someindividuals 1989,Trends fromM R.Capecchi, mice.lAdapted is,knockout Genet5:70.1

E gffl'"? ::l[T;"0?]X'J"'""' Brown mouse (NA, X-lx+) B l a c km o u s e (ala, X+lX+l 4.5-dayblastocyst

I

EIl"[';:Xl'.ffi ['Ji:;i]:il:; * A F o s t e rm o t h e r

SpecificGenesCan Be PermanentlyInactivated i n t h e G e r m L i n eo f M i c e Many of the methods for disrupting genesin yeast can be applied to genesof higher eukaryotes.Thesealteredgenescan be introduced into the germ line via homologous recombination to produce animals with a gene knockout, or simply "knockout." Knockout mice in which a specificgeneis disrupted are a powerful experimental systemfor studying mammalian development, behavior, and physiology. They also are useful in studying the molecular basisof certain human geneticdiseases. Gene-targetedknockout mice are generatedby a two-stage procedure.In the first stage,a DNA construct containing a disrupted allele of a particular target gene is introduced into embryonic stem (ES)cells.Thesecells,which are derived from the blastocyst,can be grown in culture through many generations (seeFigure 21'-7).In a small fraction of transfectedcells, the introduced DNA undergoeshomologousrecombinationwith the target gene, although recombination at nonhomologous chromosomal sitesoccurs much more frequently' To selectfor cells in which homologous gene-targetedinsertion occurs' the recombinant DNA construct introduced into ES cells needsto include two selectablemarker genes (Figure 5-40). One of thesegenes(neo'),which confersG-418 resistance,is inserted within the target gene (X), thereby disrupting it. The other selectable gene, the thymidine kinase gene from herpes simplex virus (lAHsv),confers sensitivity to ganciclovir, a cytotoxic nucleotide analog; it is inserted into the construct outside the target-genesequence.Only ES cells that undergo homologous reIo-bitr"tiott, and therefore do not incorporate tkHsv, can survivein the presenceof both G-418 and ganciclovir.In these cellsone alleleof geneX will be disrupted. In the second stage in production of knockout mice' ES cellsheterozygousfor a knockout mutation in geneX are injected into a recipient wild-type mouse blastocyst,which subsequentlyis transferred into a surrogatepseudopregnant female mouse (Figure 5-41). The resultingprogeny will be chimeras, containing tissues derived from both the transplantedES cellsand the host cells.If the ES cellsalso are ho-

Black I S.t""t chimericmicefor to wild-typeblackmice I crosses

v

germcells: All germcells: Possible a/X+ A/X+tA/X-: a/X* =r I tt cell-deriveo ProgenY s I will be brown

v

a/a, X+/X* Progenyfrom ES cell-derived germcells !| | s.re"n brown ProgenYDNA -* to identify X'lX+ heterozygotes al EI

Mate X-lX* heterozYgotes

Screen Progeny DNA to identifY lil - I l, X tx- homozYgotes

Knockoutmouse

mozygous for a visible marker trait (e.g., coat color)' then chimeric progeny in which the ES cells survived and proliferated can te identified easily.Chimeric mice are then mated with mice homozygous for another allele of the marker trait to determine if the knockout mutation is incorporated into the germ line. Finally, mating of mice' each heterozygous for the knockout allele, will produce progeny homozygous for the knockout mutation.

C E N E SI N E U K A R Y O T E S T H E F U N C T I O NO F S P E C I F I G INACTIVATING

207

loxP mouse

Cre mouse

A l l c e l l sc a r r ye n d o g e n o u sg e n e Xwith /oxPsitesflankingexon 2

Heterozygousfor gene X knockout; all cellscarry cre gene

Cell{ype-specif ic promoter

Cellsnot expressingCre

CellsexpressingCre

-4lJ@

G e n ef u n c t i o ni s n o r m a r

IoxP-Cre mouse All cells carry one copy of loxpmodified gene X, one copy of g e n eX k n o c k o u ta, n d c r e g e n e s

EXPERIMENTAL FTGURE 5-42 The loxp-Crerecombination system can knock out genes in specificcell types. A /oxpsjte is insertedon eachsideof the essential exon2 of the targetgeneX (blue)by homologousrecombination, producinga /oxpmouse.Since the /oxPsitesare in introns,they do not disruptthe functionof X T h e C r e m o u s ec a r r i e o s n e g e n eX k n o c k o u at l l e l ea n d a n i n t r o d u c e d p'l linkedto a cell-type_specific cre gene (orange)from bacteriophage promoter(yellow)The cre gene is incorporatedinto the mouse genomeby nonhomologousrecombination and doesnot affectthe

Development of knockout mice that mimic certain human diseasescan be illustrated by cystic fibrosis. By methodsdiscussedin Section5.4, the recessrve murarion that causesthis diseaseeventuallywas shown to be located in a geneknown as CFTR, which encodesa chloride chan_

rnice are currently being used as a model systemfor stud ing this geneticdiseaseand developingeffectivetherapies

S o m a t i cC e l lR e c o m b i n a t i o C n a nI n a c t i v a t e G e n e si n S p e c i f i cT i s s u e s Investigatorsoften are interestedin examining the effects o f k n o c k o u t m u t a t i o n si n a p a r t i c u l a rt i s s u eo i t h e m o u s e , at a specificstagein development,or both. However, mice carrying a germ-line knockout may have defectsin numer_ ous tissuesor die before the developmentalstageof inter_ est. To addressthis problem, mouse geneticistshave de_ v i s e d a c l e v e r t e c h n i q u et o i n a c t i v a l er a r g e r g e n e s l n 208

.

c H A p r E5R | M o L E c u L AGRE N E Tr tEcc H N t o u E S

-

FE G e n ef u n c t i o n isdisrupted

functionof othergenesln the/oxp-Cre mtcethat resultfrom crossing, Creproteinisproduced onlyin thosecellsin whichthe promoter isactiveThusthesearethe onlycellsin which recombrnation between the/oxPsites catalyzed by Creoccurs, leading to deletion of exon2 Since the otheralleleisa constitutive geneX knockout, deletion between the/oxpsitesresults in complete l o s so f f u n c t i o on f g e n e X i na l lc e l l se x p r e s s iC ng r e B yu s i n g promoters, different researchers canstudytheeffects of knockinq out geneX in various typesof cells specifictypes of somatic cells or at particular times during development This techniqueemploys site-specificDNA recombination sites(calledloxP sires)and the enzymeCre that catalyzesrecombination between them. The loxP-Cre recombination system is derived from bacteriophageP1, but this sitespecificrecombination sysremalso functions when placed in mouse cells. An essentialfeature of this technique is that expression of Cre is controlled by a cell-type-specificpromoter. In loxP-Cre mice generatedby the procedure depicted in Figure 5-42,inactivation of the geneof interest (X) occurs only in cells in which the promoter controlling the cre gene is active. An early application of this techniqueprovided strong evidencethat a particular neurotransmitterreceptor is important for learning and memory. Previouspharmacological and physiologicalstudieshad indicated that normal learning requiresthe NMDA classof glutamatereceptorsin the hippocampus,a region of the brain. But mice in which the gene encoding an NMDA receptor subunit was knocked out died neonatally,precluding analysis of the receptor's role in learning. Following the protocol in Figure 5-42, researchersgeneratedmice in which the receptorsubunit gene was inactivatedin the hippocampusbut expressedin other tissues. These mice survived to adulthood and showed

llll+ Technique Mouse Animation:creatinga Transgenic z/ar\ \\"2

DNA injected into a Pronucleusof a Mouse

miceare produced FIGURE 5-43 Transgenic > EXPERIMENTAL by randomintegrationof a foreigngeneinto the mousegerm (themale intooneof thetwo pronuclei DNAinjected line.Foreign hasa good bythe parents) andfemalehaploidnucleicontributed of the intothe chromosomes integrated of beingrandomly chance intothe recipient is integrated a transgene diploidzygoteBecause it doesnot disrupt recombination, genomeby nonhomologous l l7 . 2 7 3 1 L B r i n s t e r e, t1a9l 8 1C e n d o g e n o u s g e n[ S e se e R , e2

Injectforeign DNA intooneofthe pronuclei

Pronuclei

Fertilizedmouse egg Prlor t o f u s i o no f m a l e a n d f e m a l ep r o n u c l e i

II Transferinjectedeggs I into foster mother

learning and memory defects,confirming a role for thesereceptors in the ability of mice to encode their experiences rnto memory.

I

+

D o m i n a n t - N e g a t i vAel l e l e sC a n F u n c t i o n a l l y l n h i b i t S o m eG e n e s In diploid organisms,as noted in Section5.1, the phenotypic effect of a recessiveallele is expressedonly in homozygous individuals,whereasdominant allelesare expressedin heterozygotes.Thus an individual must carry two copies of a recessiveallele but only one copy of a dominant allele to exhibit the corresponding phenotypes. Sfe have seen how strains of mice that are homozygous for a given recessive knockout mutation can be produced by crossingindividuals that are heterozygousfor the same knockout mutation (see Figure 5-41). For experimentswith cultured animal cells, however, it is usually difficult to disrupt both copies of a genein order to produce a mutant phenotype.Moreover, the difficulty in producing strainswith both copiesof a genemutated is often compounded by the presenceof related genes of similar function that must also be inactivated in order to reveal an observablephenotype. For certain genes,the difficulties in producing homozygous knockout mutants can be avoided by use of an allele carrying a dominant-negativemutation. Theseallelesare genetically dominant; that is, they produce a mutant phenotype even in cells carrying a wild-type copy of the gene. However, unlike other types of dominant alleles,dominantnegativeallelesproduce a phenotypeequivalentto that of a loss-of-function mutatton. Useful dominant-negativealleleshave been identified for a variety of genesand can be introduced into cultured cells by transfection or into the germ line of mice or other organisms. In both cases,the introduced geneis integratedinto the genome by nonhomologous recombination. Such randomly inserted genesare called transgenes;the cells or organisms carrying them are referred to as transgenic.Transgenescarrying a dominant-negative allele usually are engineeredso that the allele is controlled by a regulatedpromoter, allowing expressionof the mutant protein in different tissuesat different times. As noted above, the random integration of exogenousDNA via nonhomologousrecombinationoccursat

o About 10-30% of offspringwill contain foreign DNA in chromosomesof a l l t h e i r t i s s u e sa n d g e r m l i n e Breed mice expressing foreign DNA to proPagate DNA in germline

@

@

a much higher frequency than insertion via homologous recombination. Becauseof this phenomenon' the production of transgenicmice is an efficient and straightforward process

GTPasesfrom an inactive GDP-bound state to an active GTP-bound state dependson their interacting with a corresponding guanine nucleotide exchangefactor (GEF)' A ,rr,.riu.r,small GTPase that permanently binds to the GEF orotein will block conversion of endogenous wild-type imall GTPasesto the active GTP-bound state' thereby inhibiting them from performing their switching function ( F i g u r e5 - 4 4 ) .

C E N E SI N E U K A R Y O T E S T H E F U N C T I O NO F S P E C I F I G INACTIVATING

(a) Cellsexpressingonly wild-type allelesof a small GTPase

Inactive

j

fGo' \-/ Wildtype

(b) Cellsexpressingboth wild-type allelesand a dominant-negativeallele

D o m i n a n t - n e guve a mutant

FIGURE 5-44 Inactivationof the functionof a wild-type GTPase by the actionof a dominant-negative mutantallele. (a)Small(monomeric) (purple) GTPases areactivated by their interaction with a guaninenucleotide exchange factor(GEF), which catalyzes the exchange of GDpfor GTp(b)Introduction of a dominant-negative alleleof a smallGTpase geneintocultured cellsor transgenic animals leadsto expression of a mutantGTpase that binds to andinactivates the GEFAsa result, endogenous wild-type copies of thesamesmallGTPase aretrappedin the inactive GDp-bound stateA singledominant-negative allelethuscauses a loss-offunctionphenotype in heterozygotes similar to thatseenin homozygotes carrying two recessive loss-of-f unctionalleles

R N AI n t e r f e r e n c e C a u s e sG e n eI n a c t i v a t i o nb y Destroyingthe CorrespondingmRNA A recently discoveredphenomenon known as RNA interference (RNAi) is perhaps the mosr straightforward method to inhibir the function of specificgenes.This approach is technically simpler than the methods described above for disrupting genes.First observed in the roundworm C. elegans,RNAi refers to rhe ability of doublestranded RNA to block expression of its corresponding single-strandedmRNA but not that of mRNAs with a difl ferent sequence. As describedin Chapter 8, the phenomenon of RNAi rests on the general ability of eukaryotic cells to cleave double-strandedRNA inro short (23-nt) double-stranded segmentsknown as small inhibitory RNA (siRNA). The RNA endonuclease that catalyzesthis reaction, known as

tween one strand of the siRNA and its complementaryse_ quenceon the target nRNA; subsequently,specificnucle_ asesin the RISC complex then cleave the mRNA/siRNA hybrid. This model accounts for the specificity of RNAi, since it dependson basepairing, and for its potency in si_ lencing gene function, since the complem entary mRNA is 21O

.

cHAprER 5

|

M o L E c u L AG R E N E T tr cE c H N t e u E s

permanently destroyed by nucleolytic degradation. The normal function of both Dicer and RISC is to allow for gene regulation by small endogenous RNA molecules known as micro RNAs (miRNAs). Researchersexploit the micro RNA pathway for intentional silencing of a gene of interest by using either of two generalmethods for generatingsiRNAs of defined sequence. In the first method a double-strandedRNA corresponding to the target genesequenceis produced by in vitro transcription of both senseand antisensecopiesofthis sequence(Figure5-45a). This dsRNA is injected into the gonad of an adult worm. where it is converted to siRNA by Dicer in the developing embryos. In conjunction with the RISC complex, the siRNA molecules causethe corresponding mRNA molecules to be destroyedrapidly. The resulting worms display a phenotype similar to the one that would result from disruption of the corresponding gene itself. In some cases,entry of just a few moleculesof a particular dsRNA into a cell is sufficient to inactivate many copies of the corresponding mRNA. Figure 5-45b illustrates the ability of an injected dsRNA to interfere with production of the corresponding endogenousmRNA in C. elegansembryos.In this experiment, the mRNA levels in embryos were determined by incubating the embryos with a fluorescently labeled probe specific for the mRNA of interest. This technique,in situ hybridization, is useful in assayingexpressionof a particular mRNA in cells and tissuesections. The second method is to produce a specific doublestranded RNA in vivo. An efficient way ro do this is to expressa synthetic gene thar is designedto contain tandem segmentsof both senseand anti-sensesequencescorresDonding to the target gene (Figure 5-45c). \X/henthis gene is transcribed, a double-strandedRNA "hairpin" srructure forms, known as small hairpin RNA, or shRNA. The shRNA will then be cleaved by Dicer to form siRNA molecules. The lentiviral expressionvectors are particularly useful for introducing synrhericgenes for the expressionof shRNA constructsinto animal cells. Both RNAi methods lend themselves to systematic studiesto inactivateeach of the known genesin an organism and to observewhat goeswrong. For example, in initial studies with C. elegans,RNA interferencewith 16,700 genes(about 86 percent of the genome)yielded 1722 visibly abnormal phenotypes.The geneswhose functional inactivation causesparticular abnormal phenotypescan be grouped into sets;each member of a set presumably controls the same signals or events.The regulatory relations between the genes in the set-for example, the genes that control muscledevelopment-can then be worked out. Other organismsin which RNAi-mediated geneinactivation has been successfulinclude Drosophila, many kinds of plants, zebra fish, the frog Xenopus, and mice, and are now the subjects of large-scaleRNAi screens.For example, lentiviral vectors have been designedto inactivate by RNAi more than 10,000 different genes expressedin cultured mammalian cells. The function of the inactivated genescan be inferred from defects in growth or morphology of cell clones transfectedwith lentiviral vectors.

fi| ,oo."rt: RNAInterference (a) In vitro production of double-stranded RNA Antisensetranscript

--\(-

Injected

Noninjected

(c) In vivo production of double-stranded RNA

(RNA|)can 5-45 RNAinterference FIGURE < EXPERIMENTAL functionallyinactivategenesin C.elegansand other for RNA(dsRNA) of double-stranded organisms.(a)Invitroproduction gene, the gene of sequence The coding target RNA|of a specific DNA,is of genomic fromeithera cDNAcloneor a segment derived to a strong vectoradjacent in a plasmid placedin two orientations in vitrousingRNA of bothconstructs promoterTranscription in yields manyRNAcoptes triphosphates polymerase andribonucleoside (identical or sequence) mRNA with the orientation thesense these conditions, orientationUndersuitable antisense complementary to formdsRNAWhenthe willhybridize RNAmolecules complementary by DicerintosiRNAs(b) intocells,it iscleaved isinjected dsRNA by RNA|(seethe worm embryos in of mex3RNAexpression lnhibition was (Left) RNA in embryos mex3 of Expression mechanism) for the text for thismRNA,that with a probespecific by in situhybridization assayed product(Rtgtht) a colored(purple) thatproduces is,linkedto an enzyme mex3 double-stranded with froma worminjected Theembryoderived indicated by mRNA, as mex3 no endogenous produces little or mRNA embryois=50 Fm in length of color.Eachfour-cell-stage theabsence viaan engineered RNAoccurs (c)Invivoproduction of double-stranded geneconstruct isa intocellsThesynthetic directly plasmid introduced of the sequences and antisense sense of both tandemarrangement RNA smallhairpin double-stranded targetgeneWhenit istranscribed, (b) by Dicerto formsiRNAlPart iscleaved TheshRNA forms(shRNA) Nature 391:806 etal. 1998, l fromA Fire

Sensetranscript

r The /oxP-Cre recombination system permits production of mice in which a geneis knocked out in a specifictissue.

siRNA 2+

Inactivating the Function of SpecificGenes in Eukaryotes r Once a gene has beencloned, important clues about its normal function in vivo can be deducedfrom the observed phenotypic effectsof mutating the gene. r Genescan be disrupted in yeast by inserting a selectable marker geneinto one allele of a wild-rype genevia homologous recombination, producing a heterozygousmutant. \7hen such a heterozygoteis sporulated, disruption of an essential genewill producetwo nonviablehaploid spores(Figure5-39). r A yeastgenecan be inactivatedin a controlledmannerby using the GALI promoter to shut off transcription of a genewhen cellsare transferredto glucosemedium.

r In the production of transgeniccells or organisms,exogenous DNA is integrated into the host genome by nonhomologous recombination (seeFigure 5-43). Introduction of a dominant-negativeallele in this way can functionally inactivate a genewithout altering its sequence. r In many organisms,including the roundworm C. elegans, double-stranded RNA triggers destruction of the all the mRNA moleculeswith the samesequence(seeFigure 5-45). This phenomenon, known as RNAI (RNA interference), provides a specificand potent means of functionally inactivating geneswithout altering their structure.

As the examplesin this chapter and throughout the book illustrate, genetic analysis is the foundation of our understanding of many fundamental processesin cell biology. By examining the phenotypic consequencesof mutations that inactivate a particular gene' geneticistsare able to connect knowledge about the sequence'structure' and biochemical

r In mice, modified genescan be incorporated into the germ line at their original genomic location by homologous recombination,producingknockouts (seeFigures5-40 and 5-41).Mouse knockoutscan providemodelsfor human genetic diseases such as cysticfibrosis. P E R S P E C T I VF EO S RT H E F U T U R E

211

Although scientistscontinue to use this classicalgenetic approachto dissectfundamentalcellular processes and biochemical pathways, the availability of iomplete genomic sequenceinformation for most of the common experimental organisms has fundamentally changed the way genetic experiments are conducted. Using various computational methods, scientistshave identified the protein-coding gene sequences in most experimentalorganismsincludingE. coli, yeast, C. elegans,Drosophila, Arabidopsis, mouse, and humans.The genesequences, in turn, revealthe primary amino acid sequenceof the encodedprotein products, providing us with a nearly complete list of the proteins found in each of the major experimentalorganisms. The approach taken by most researchershas thus shifted from discoveringnew genesand proteins to discoveringthe functions of genesand proteins whose sequencesare already known. Once an interestinggenehas beenidentified,genomic sequenceinformation greatly speedssubsequentgeneticmanipulations of the gene,including its designedinactivation, to learn more about its function. Already sets of vectors for RNAi inactivation of most defined senesin the nematode C. elegansnow allow efficientgeneric,ir..n, to be performed in this multicellular organism.Thesemethodsare now being applied to largecollectionsof genesin cultured mammalian cells and in the near future either RNAi or knockout methodswill have beenusedto inactivateeverygenein the mouse. In the past, a scientistmight spend many years studying only a single gene, but nowadays scientistscommonly study whole setsof genesar once. For example,with DNA microarrays the level of expressionof all genesin an organismcan be measuredalmost as easily as the expressionof a singlegene. One of the great challengesfacing geneticistsin the twentyfirst century will be to exploit the vast amount of available data on the function and regulation of individual genesto understandhow groups of genesare organizedto form complex biochemicalpathways and regulatory nerworks.

KeyTerms alleles 166

linkage175

ctone I /')

tlntation 765 Northern blotting 192 phenotype155

|

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complementary DNAs (cDNAs) 181 complementation183 DNA cloning 126 DNA library 179 DNA microarray 192 dominant L66 geneknockoul'207 genomics129 genotype166 heterozygous166 homozygous165 hybridization 181 in situ hybridizatton 192 212

.

c H A p r EsR

plasmids178 polymerasechain r e a c t i o n( P C R )1 8 8 p r o b e s1 8 1 recessive165 recombinantDNA 126 recombination175 restriction enzymes176 RNA interference (RNAi)210 segregation167 Southern blotting 191 MOLECULAG RENETIC TECHNIQUES

temperature-sensrtrve mutations 170

transgenes209 vector 1-76

transfection 196 transformationL78

Review the Concepts 1, Genetic mutations can provide insights into the mechanisms of complex cellular or developmentalprocesses.How might your analysisof a geneticmutation be different depending on whether a particular mutation is recessiveor dominant? 2. Give an example of how and why temperature-sensitive mutations might be usedto study the function of essentialgenes. 3. Describehow complementation analysiscan be used to reveal whether two mutations are in the sameor in different genes.Explain why complementation analysiswill not work with dominant mutations. 4, Compare the different usesof suppressorand synthetic lethal mutations in geneticanalysis. 5. Restriction enzymesand DNA ligaseplay essentialroles in DNA cloning. How is it that a bacterium that produces a restriction enzyme does not cut its own DNA? Describe some general features of restriction enzymesites.ril/hat are the three types of DNA ends that can be generatedafter cutting DNA with restriction enzymes?\{hat reaction is catalyzedby DNA ligase? 6. Bacterial plasmids often serve as cloning vectors. Describethe essentialfeaturesof a plasmid vector. Sfhat are the advantagesand applicationsof plasmidsas cloning vectors? 7. A DNA library is a collectionof clones,eachcontaining a different fragment of DNA, inserted into a cloning vector. What is the differencebetweena cDNA and a genomic DNA library? How can you use hybridization or expressionto screen a library for a specific gene? How many different oligonucleotideprimers would need to be synthesizedas probes to screena library for the gene encoding the peptide Met-Pro-Glu-Phe-Tyr? 8. In 1993, Kerry Mullis won rhe Nobel prize in chemistry for his invention of the PCR process.Describethe three steps in each cycle of a PCR reaction. $fhy was the discoveryof a thermostableDNA polymerase(e.g.,Taq polymerase)so important for the developmentof PCR? 9. Southernand Northern blotting are powerful tools in molecular biology basedon hybridization of nucleic acids. How are these techniques the same? How do they differ? Give some specific applications for each blotting technique. 10. A number of foreign proteins have been expressedin bacterial and mammalian cells. Describe the essential featuresof a recombinantplasmid that are required for expressionof a foreign gene.How can you modify the foreign protein to facilitate its purification? What is the advantage of expressinga protein in mammalian cellsversusbacteria? 11. What is a DNA microarray? How are DNA microarrays usedfor studying geneexpression?How do experimentswith microarrays differ from Northern blotting experiments?

12. In determining the identity of the protein that corresponds to a newly discoveredgene, it often helps to know the pattern of tissueexpressionfor that gene.For example, researchers have found that a genecalledSERPINA6 is expressedin the liver, kidney, and pancreasbut not in other tissues.!7hat techniques might researchersuse to find out which tissuesexpressa particular gene?

b. A wild-type yeastcDNA library, preparedin a plasmid that contains the wild-type URA3* gene' is used to transformX cells,which are then culturedas indicated.Each black spot below representsa singleclone growing on a petri '$7hat plate. are the molecular differencesbetweenthe clones growing on the two plates?How can theseresults be used to identify the geneencodingX?

13. DNA polymorphisms can be used as DNA markers. Describethe differencesbetweenRFLR SNR and SSRpolymorphisms.How can thesemarkersbe usedfor DNA mapping studies? 14. How can linkage disequilibrium mapping sometimcs provide a much higher resolutionof genelocation than classicallinkagemapping? 15. Genetic linkage studies can usually only roughly locate the chromosomalposition of a "disease"gene.How can expression analysis and DNA sequenceanalysis help locate a diseasegenewithin the region identified by linkage mapping? 16. Gene targetingby siRNA techniquesexploits a normal micro RNA pathway that is found in all metazoansbut not in simpler eukaryotessuch as yeast.\7hat are the roles of Dicer and RISC in this pathway? 77. The ability to selectivelymodify the genome in the mousehas revolutionizedmousegenetics.Outline the procedure for generatinga knockout mouse at a specificgenetic locus.How can the loxP-Cresystembe usedto conditionally knock out a gene?\Vhat is an important medicalapplication of knockout mice? 18. Two methods for functionally inactivating a gene withmuout alteringthe genesequenceare by dominant-negative tations and RNA interference(RNAi). Describehow each method can inhibit expressionof a gene.

A culture of yeast that requiresuracil for growth (ura3-) was mutagenized,and two mutant colonies,X and Y, have been isolated.Mating type a cells of mutant X are mated with mating type o cellsof mutant Y to form diploid cells. The parental (ura3 ), X, Y, and diploid cells are streaked onto agar platescontaininguracil and incubatedat 23 "C or 32'C. Cell growth was monitored by the formation of colonieson the culture platesas shown in the figure below. Denotesgrowth of cells

ura3 '/'

ura

w

Parental X

Growthat 23'C

Parental X PCR

d. A construct of the wild-type geneX is engineeredto encode a fusion protein in which the green fluorescentprotein (GFP) is presentat the N-terminus (GFP-X) or the Cterminus (X-GFP) of protein X. Both constructs'presenton a URA3* plasmid, are used to transform X cells grown in the absenceof uracil. The transformants are then monitored for growth at 32oC, shown below at the left. At the right are typical fluorescentimagesof X-GFP and GFP-X cells grown at 23 "C in which green denotesthe presenceof green fluorescentprotein. What is a reasonableexplanation for growth o f G F P - X b u t n o t X - G F P c e l l sa t 3 2 ' C ?

\X \

\

/,

G r o w t ha t 3 2 ' C o n m e d i al a c k i n gu r a c i l

c. DNA is extracted from the parental cells, from X cells, and from Y cells and digested with a restriction enzyme.The digests are analyzedby Southern blot analysis, shown at the left below, using a probe obtained from the gene encoding X. In addition, PCR primers are used to amplify the gene encoding X in both the parental and the X cells.The primersare complementaryto regionsof DNA just external to the geneencoding X. The PCR results are shown in the gel at the right. What can be deducedabout the mutation in the X genefrom thesedata?

Southern

Analyzethe Data

Diploid

G r o w t ha t 2 3 ' C o n m e d i al a c k i n gu r a c i l

Diploid

G F P - Xc e l l

/, G r o w t ha t 3 2 ' C X-GFP

What can be deducedabout mutants X and Y from

a. the data provided?

Growth at 32 "C

G F Pl o c a l i z a t i oinn c e l l s grown at 23 'C THE DATA ANALYZE

O

213

e. Haploid offspring of the diploid cells from part (a) above are generated.XY double mutants constitute 114 of theseoffspring.Haploid X cells,Y cells,and XY cellsin liquid culture are synchronizedat a stagejust prior to budding and then are shifted from 23'C to 32 oC. Examination of the cells 24 hours later revealsthat X cellsare arresredwith small buds, Y cellsare arrestedwith large buds, and XY cellsare arrested with small buds.\Whatis the relationshipbetweenX and Y?

References Genetic Analysis of Mutations to ldentify and Study Genes Adams,A. E. M., D. Botstein,and D. B. Drubin. 1989.A yeast actin-bindingprotein is encodedby sac6,a genefound by suppression of an actin muration. Science243:231. Griffiths, A. G. F., et al. 2000. An Introduction to Genetic Analysis,Tth ed. W. H. Freemanand Company. Guarente,L.1993. Synthericenhancement in geneinteraction:a genetictoof comesof age.TrendsCenet.g:362-3o6. Hartwell, L. H. 1967.Macromolecularsynthesis of temperature-sensitive mutanrs of yeast.J. Bacteriol. 93:1662. Hartwell, L.H. 1974. Geneticcontrol of the cell divisioncycle in yeast.Science183:46. Niisslein-Volhard, C., and E.'Wieschaus. 1980.Mutations affectins segmentnumber and polariryin Drosophila.Nature 287:795-801. Simon,M. A., er al. 1991. Rasl and a putativeguaninenucleotide exchangefactor perform crucialstepsin signalingby the sevenless protein tyrosinekinase.Cell 67:701-776. Tong,A. H., et al. 2001. Systematicgeneticanalysiswith ordered arraysof yeastdeletionmurants.Science294:2364)368. DNA Cloning and Characterization Ausubel,F. M., et al.2002. Current Protocolsin Molecular Biology.Vlley. Gubler,U., and B. J. Hoffman. 1983.A simpleand very efficient method for generatingcDNA libraries. Gene 25:263-289. Han, J. H., C. Stratowa,and W. I. Rutter.1987.Isolationof fulllengthputativerat lysophospholipase cDNA usingimprovedmethods for mRNA isolationand cDNA cloning.Biochem.26:.161.7-1632. Itakura,K., J.J. Rossi,and R. B. Wallace.1984.Synthesis and use of syntheticoligonucleotides.Ann. Reu.Biochem. 53:323-356. Maniatis, T., et al. 1978.The isolation of structural senesfrom libraries of eucaryoticDNA. Cel/ 15:687-701. Nasmyth,K. A., and S. I. Reed.1980.Isolationof senesby complementationin yeast:molecularcloning of a cell-Jyclegene. Proc. Nat'l Acad. Sci.USA 77:21.19-2123. Nathans,D., and H. O. Smith. 1975.Restrictionendonucleases in the analysisand restructuringof DNA molecules.Ann. Reu.Biochem. 44:273193. Roberts,R. J., and D. Macelis.1997.REBAsE-restricrion enzymesand methylases.Nacl. Acids Res.25:248-262. Information on accessinga continuouslyupdateddatabaseon restrictionand modification enzymesat http://www.neb.com/rebase.

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M O L E C U L AG R E N E T TTCE C H N T Q U E S

Using Cloned DNA Fragments to Study Gene Expression Andrews, A.T. 1986. Electrophoresis,2d ed. Oxford University Press. Erlich, H., ed. 1.992.PCR Technology:Principlesand Applicationsfor DNA Amplification. W. H. Freemanand Company. Pellicer,A., M. Wigler, R. Axel, and S. Silverstein.1978. The transfer and stableintegration of rhe HSV thymidine kinasegene into mousecells.Cel/ 4t:1,33-1{1,. Saiki,R. K., et al. 1988. Primer-directed enzymaticamplification of DNA with a thermostableDNA polymerase.Science239:487491. Sanger,F. 1981. Determinationof nucleotidesequences in DNA. Science214:720 5-'1210. Souza,L. M., et al. 1986.Recombinanthuman granulocyte-colony stimulating factor: effectson normal and leukemic myeloid cells. Science232:61-65. 'Wahl, G. M., J. L. Meinkoth, and A. R. Kimmel. 1987. Northern and Southern6lots. Metb. Enzymol. 152:572-581. Wallace,R. B., et al. 1,987.The useof syntheticoligonucleotides as hybridizationprobes.II: Hybridization of oiigonucleotides of mixed sequence to rabbit B-globinDNA. Nzcl. Acids Res.9:879-887. ldentifying and Locating Human Disease Genes Botstein,D., et al. 1980. Constructionof a geneticlinkage map in man using restriction fragmentlength polymorphisms.Az. /. Genet.32:31.4-331.. Donis-Keller,H., et al. 1,987.A geneticlinkage map of the human genome.Cell 5l:319-337. Hartwell, et al. 2000. Genetics:From Genesto Genomes. McGraw-Hill. Hastbacka,T., et al. 1994.The diastrophicdysplasiagene encodesa novel sulfatetransporter:positional cloning by finestructurelinkage disequilibriummapping. Cell 781.073. Orita, M., et al. 1989. Rapid and sensitivedetecrionof point mutations and DNA polymorphismsusing the polymerasechain reaction. Genomics5:874. Tabor,H. K., N.J. Risch,and R. M. Myers.2002. Opinion: candidate-gene approachesfor studyingcomplex generictrairs: practicalconsiderations.Nat. Reu.Genet.3:391,-397. Inactivating the Function of Specific Genes in Eukaryotes Capecchi,M. R. 1989. Altering the genomeby homologousrecombination. Science 244 :'1,288-1,292. Deshaies,R. J., et al. 1988. A subfamily of stressproteins facili, tatestranslocationof secretoryand mitochondrial precursor polypeptides.Nature 332:800-805. Fire, A., et al. 1,998.Porentand specificgeneticinterferenceby double-strandedRNA in Caenorhabditiselegans.Nature 391:806-8'1.1. Gu, H., et al. 1994. Delerion of a DNA polymerasebeta gene segmentin T cellsusing cell type-specificgenetargeting.Science 265:L03-106. Zamore, P. D., T. Tuschl,P.A. Sharp,and D. P. Bartel.2000. RNAi: double-strandedRNA directsthe ATP-dependentcleavageof mRNA at 21 to 23 nucleotideinrervals.Cell 101:25-33. Zimmer, A. 1992. Manipulating the genomeby homorogous recombinationin embryonic stem cells.Ann. Reu.Neurosci. 15:115

CHAPTER

GENES, AND GENOMICS, CHROMOSOMES ThesebrightlycoloredRxFISH-painted chromosomes areboth anomalies and in beautifuland usefulin revealing chromosome of Clinical comparingkaryotypes of differentspecies[@Department Researchers, Incl Cytogenetics, Addenbrookes Hospital/Photo

I n previous chapterswe learnedhow the structure and comI position of proteins allow them to perform a wide variety I of cellular functions. We also examined another vital component of cells, the nucleic acids, and the processby which information encoded in the sequenceof DNA is translated into protein. In this chapter,our focus again is on DNA and proteins as we consider the characteristicsof eukaryotic nuclear and organellar genomes:the features of genesand the other DNA sequencethat comprise the genome, and how this DNA is structured and organized by proteins within the cell. By the beginning of the twenty-first century, molecular biologists had completed sequencingthe entire genomesof hundredsof viruses,scoresof bacteria,and one unicellular eukaryote, the budding yeast S. cereuisiae.In addition, the vast majority of the genome sequenceis also known for the fission yeast S. pombe, and severalmulticellular eukaryotes including the roundworm C. elegans, the fruit fly D. melanogastel,mice, and humans. Detailed analysis of these sequencingdata has revealedinsights into genome organtzation and genefunction. It has allowed researchersto identify previously unknown genesand to estimatethe total number of protein-coding genesencoded in each genome. Comparisons betweengenesequencesoften provide insight into possible functions of newly identified genes. Comparisons of genomesequenceand organizationbetweenspeciesalso help us understand the evolution of organisms. Surprisingly,DNA sequencingrevealedthat a large portion of the genomesof higher eukaryotes does not encode mRNAs or any other RNAs required by the organism. Remarkably, such noncoding DNA constitutes :98.5 percent

of human chromosomal DNA! The noncoding DNA in multicellular organisms contains many regions that are similar but not identical. Variations within some stretchesof this repetitious DNA between individuals are so great that every person can be distinguishedby a DNA "fingerprint" based on these sequencevariations. Moreover, some repetitious DNA sequencesare not found in the same positions in the genomesof different individuals of the same species.At one "junk time, all noncoding DNA was collectively termed 'We now DNA" and was considered to serve no purpose. understandthe evolutionary basisof all this extra DNA, and the variation in location of certain sequencesbetween

OUTLINE 6.1

EukaryoticGene Structure

217

6.2

C h r o m o s o m aOl r g a n i z a t i o no f G e n e s a n d N o n c o d i n gD N A

223

5.3

(Mobile) DNA Elements Transposable

226

6.4

O r g a n e l l eD N A s

236

5.5

G e n o m i c sG: e n o m e - w i d eA n a l y s i so f G e n e Structureand ExPression

6.6

StructuralOrganizationof Eukaryotic Chromosomes

6.7

M o r p h o l o g ya n d F u n c t i o n aEl l e m e n t s of EukaryoticChromosomes

215

individuals. Cellular genomesharbor symbiotic transposable (mobile) DNA elements,sequencesthat can copy themselves and move throughout the genome.Although transposable DNA elementsseemro have little funcrion in the life cycleof an individual organism, over evolutionary time they have shapedour genomesand contributedto the rapid evolution of multicellularorganisms. In higher eukaryores,DNA regions encoding proteins or functional RNAs-that is, genes-lie amidst this expanseof apparently nonfunctional DNA. In addition ro the nonfunctional DNA between genes,noncoding introns are common within genesof multicellularplants and animals.Sequencing of the same protein-coding gene in a variety of eukaryotic specieshas shown that evolutionary pressureselectsfor maintenanceof relativelysimilar sequences in the coding reglons,or exons.In contrast,wide sequence variation,evenincludingtotal loss,occursamon€lintrons,suggesting that most intron sequenceshave little functional significance.However, as we shall see,although most of the DNA sequenceof introns is not functional,the existenceof introns has favored the evolution of multidomain proteins that are common ln highereukaryotes.It also allowed the rapid evolutionof proteinswith new combinationsof functional