AGENETICSWITCHTHIRDEDITION

PhageLambdaRevisited

MarkPtashneMemorialSloanKetteringCancerCenterNewYork

COLDSPRINGHARBORLABORATORYPRESSColdSpringHarbor,NewYorkhttp://www.cshlpress.com

AGENETICSWITCHPhageLambdaRevisitedThirdEdition

Allrightsreserved.2004byColdSpringHarborLaboratoryPress,ColdSpringHarbor,NewYorkPrintedintheUnitedStatesofAmerica,ISBN978-087969716-7

PublisherJohnInglisAcquisitionEditorsAlexanderGannandJohnInglisDevelopmentalEditorAlexanderGannProjectCoordinatorMarylizDickersonProductionEditorMelissaFreyDesktopEditorSusanSchaeferProductionManagerandInteriorDesignerDeniseWeissCoverDesignerMikeAlbano

LibraryofCongressCataloging-in-PublicationDataPtashne,Mark.Ageneticswitch:phagelambdarevisited/byMarkPtashne.--3rded.

p.;cm.Includesbibliographicalreferencesandindex.ISBN0-87969-716-4(alk.paper)

1.Bacteriophagelambda.2.Geneticregulation.3.Viralgenetics.[DNLM:1.Bacteriophagelambda--genetics.2.GeneExpressionRegulation.3.Genes,Viral.4.Lysogeny.5.RepressorProteins.QW161.5.C6P975g2004]I.Title.QR342.P832004579.296--dc22

2004000803

109876543

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ThisbookisdedicatedtomyparentsandtoMattMeselson

CONTENTSPrefacetotheThirdEditionPrefacetotheFirstEdition

INTRODUCTION

CHAPTERONETHEMASTERELEMENTSOFCONTROL

ComponentsoftheSwitchDNARNAPolymeraseTheRepressorCroTheActionofRepressorandCroNegativeControlPositiveControlCooperativityofRepressorBindingInductionFlippingtheSwitchCooperativitySwitchStabilityandSensitivityTheEffectofAutoregulationOtherCases

CHAPTERTWOPROTEIN-DNAINTERACTIONSANDGENECONTROL

TheOperatorRepressorCro

AminoAcid-BasePairInteractionsThePromoterGeneControl

CHAPTERTHREECONTROLCIRCUITSSETTINGTHESWITCH

ABriefOverviewofGrowthTheGeneticMapCircularizationGeneExpressionIntegrationControlofTranscriptionVeryEarlyEarlyLateLyticLateLysogenicTheDecisionControlofIntegrationandExcisionCase1EstablishingLysogenyCase2LyticGrowthCase3InductionOtherPhagesTheSOSResponsePathwaysandCellDevelopmentRegulatoryGenesSwitchesPatternsofGeneExpression

CHAPTERFOURHOWDOWEKNOWTHEKEYEXPERIMENTS

TheRepressorIdeaClearandVirulentMutantsObservationsExplanationImmunityandHeteroimmunityObservationsExplanationAsymmetryinBacterialMatingObservationsExplanationTheRepressorProblemintheEarly1960sRepressorIsolationandDNABindingMakingMoreRepressorTheClaimsofChaptersOneandTwoTherepressoriscomposedoftwoglobulardomainsheld

togetherbyalinkerofsome40aminoacidsTherepressordimerizes,largelythroughinteractionbetween

itscarboxyldomainsArepressordimerbinds,throughitsaminodomains,toa17

basepairoperatorsiteAsingleoperatorsitebindsonedimerofrepressorDimersformbeforeDNAbindingTheaminodomainscontactDNATherearethree17basepairrepressorbindingsitesinthe

rightoperator.AteachsiterepressorandCrobindalongthesamefaceofthehelix

ChemicalprobesOperatormutationsBindingtosupercoiledandlinearDNARepressorbindstothreesitesinORwithalternatepairwise

cooperativity.Thecooperativityismediatedbyinteractionsbetweencarboxyldomainsofadjacentdimers

InalysogenrepressoristypicallyboundtoOR1andOR2.TheboundrepressorsturnoffrightwardtranscriptionofcroandstimulateleftwardtranscriptionofcI.Athigherconcentrations,repressorbindstoOR3toturnofftranscriptionofcI

CrobindsfirsttoOR3,thentoOR1andOR2,therebyfirstturningoffPRM,thenPR

SomebackgroundaboutCroCroinvivoCroinvitroRecAcleavesrepressortotriggerinductionWhenCroisboundatOR3theswitchisthrownRepressorandCrobindtotheoperatorasshowninFigures

2.6,2.8,2.10,and2.11CrystallographyThehelixswapexperimentSpecificaminoacid-basepaircontactsTheroleofthearmofrepressorRepressoractivatestranscriptionofcIbybindingtoOR2and

contactingpolymerasewithitsaminodomainPositivecontrolmutantsPositivecontrolinvitroConclusion

CHAPTERFIVE2004:NEWDEVELOPMENTS

1.Long-rangeCooperativityandRepressionofPRMAnOctamerofRepressorBindsORandOLAutonegativeRegulationofRepressorSynthesisHowDoWeKnowLong-rangeInteractionsandRepressionofPRLong-rangeInteractionsandRepressionofPRMActivationandRepressionofPRMRepressorStructure2.PositiveControl(ActivationofTranscription)PolymeraseandPromoterTheMechanismofActivationHowDoWeKnowActivatingRegionVariantsASuppressorofapcMutantCrystallographyActivatorBypassChangingActivatingRegionsandTargetContext3.TheStructureoftheRepressorMonomerandthe

MechanismofRepressorCleavageHowDoWeKnow4.EvolvingtheSwitchChangingtheAffinitiesofSitesinORforRepressorEliminatingPositiveControlEliminatingCooperativitybetweenDNA-bindingDimers5.CIIandtheDecision

APPENDIXONEDESIGNINGANEFFICIENTDNA-BINDINGPROTEIN

APPENDIXTWOSTRONGANDWEAKINTERACTIONS

Index

PREFACETOTHETHIRDEDITION

Biologistswork on systems that have evolved.This gives ushopethatanygivencasecanbeunderstoodreductively.Naturebuiltthesysteminsteps,eachstepmakinganimprovementonthe previous version and so, this line of thought goes, theinvestigatorcantakeitapart,studyitinbits,and,perhaps,seehow it all fits together. These notions have proved apt instudyinggeneregulationinthebacterialviruslambda.

Thefirsteditionofthistext(publishedin1986)describeda series of protein-protein and protein-DNA interactions thateffect two alternative patterns of gene expression in phagelambda. This genetic switch (as we called it) ensures anefficientchangefromonepatterntotheotherinresponsetoanenvironmentalsignal.

Thisneweditionispromptedbydiscoveries(noneduetomy efforts) that add to, rather than reformulate, the earlierstory.Themoststrikingofthesedescribesinteractionsbetweenproteinsbinding towidelyseparatedsitesonDNA,andshowshow these interactions make the switch even more efficientthan we had thought. Others probe more deeply than before,showing us, in clear molecular terms, how an activator oftranscriptionworks.Otherexperimentsbegintodissecthowthecomplex lambdageneregulatorynetworkmighthaveevolved,and still another reveals an enzymatic function carried by therepressoritself.

These new developments are described here in a chapter(Chapter 5) added to a reprint, only slightlymodified, of thefirst edition. Those already familiar with the originaldescriptionoftheswitchcanlearnaboutthesedevelopmentsbygoing directly to the new chapter; others can learn thewholestory by reading the complete book as it now stands. Theextendeddiscussionofgeneregulationineukaryotes,addedaspart of the second edition but omitted here, has beenreformulatedandplaced ina largercontext in thebookGenesand Signals (Ptashne and Gann 2002 Cold Spring HarborLaboratory Press). The preface to the second edition has alsobeenomittedhere.

The new chapter illustrates, as did the original text, thatuncoveringinteractionsbetweencomponentsoftheswitch,andassessing their biological significance, required combininggenetic with biochemical studies, and in some cases withbiophysical and structural studies as well. Given how weaksome of the interactions arefor example, those mediatingcooperativebindingofproteinstoDNAitishardtoseehowany other approach would have worked. Our picture of theswitch has been formulated by studying the componentreactions in isolationandthenpiecingthemtogether.Thefactthat a step-wise approach has produced a picture of such(seeming)completenessandcoherence is in itself (itseemstome)remarkable.

Strictly speaking, this book was, and remains, mistitledAn Epigenetic Switch would be more appropriate. Anepigenetic change in the state of gene expression is one thatpersistsintheabsenceoftheoriginalsignal(orevent),andthatinvolves no change in DNA sequence. The maintenance of

lysogenyovermanycell divisions following its establishment(seeChapter3)isaclassicexampleofepigenesis.

Thereareimportantmattersofgeneregulationinlambda,currentlyunderstudy,thatIdonotdiscussinthenewchapter.Themechanisms of action of the anti-terminators N andQ(introducedinChapter3)aretwoexamples.Systemsbiologistshave applied quantitativemodeling approaches to this or thataspectofthelambdalifestylesandthetransitionbetweenthem,but I have not attempted to review these matters either.Referencestosomeofthesestudiesaretobefoundat theendofChapter5.

All the figures from this book (along with those fromGenes and Signals) are available on the Web Sitewww.genesandsignals.com.

Once again generous people have come to my rescue,offering facts, figures, advice, and admonishments. I want tothankparticularlyAlexGann,whoseencouragementandadviceateverystepwascrucial,aswellasSethDarst,IanDodd,AnnHochschild, Deepti Jain, Leemor Joshua-Tor,Mitchell Lewis,John Little, and Keith Shearwin, each of whom contributedfigures or unpublished results, commented extensively on thetext,orboth.IalsothankAlanCampbell,SimonDove,RichardEbright,BarryEgan,DrewEndy,LennyGuarente,BarryHonig,Sandy Johnson, Tom Laue, Wendell Lim, Richard Losick,DavidSenear,RichardTreisman,andJoseVilar,eachofwhomalsohelpedmethinkcertainthingsthrough.MaryJoWrightstypingandorganizingsavedthedayonmorethanoneoccasion.HansNeuhart accurately redrew theold figures aswell as thenew ones, lickety-split, while the efforts of Denise Weiss,Melissa Frey, and Susan Schaefer at CSHL Press were

invaluableinputtingthebooktogether.MARKPTASHNE

NewYork,NYJanuary2004

PREFACETOTHEFIRSTEDITION

This book is about one of natures simplest organismsavirusthatgrowsonabacterium.Itdescribestheresultsofsome25 years of research on the question of how the virus calledlambda()usesitsgenesitsDNAtodirectitsgrowth.

Why has somuch effort been expended on studying onevirus?Thisisafairquestion...afterall,everycaseinbiologyisat least partly accidental and special. The workings of everyorganismhavebeendeterminedbyitsevolutionaryhistory,andthe precise descriptionwe give of a process in one organismwillprobablynotapplyindetailtoanother.Theansweristobefound in the context of the fundamental biological processcalleddevelopment.

Briefly put, the issue is as follows: all cells of a givenindividual organism inherit the same set of blueprints in theform of DNA molecules. But as a higher organism developsfrom a fertilized egg a striking variety of different cell typesemerges. Underlying the process of development is theselectiveuseofgenes,thephenomenonwecallgeneregulation.Atvariousstages,dependinginpartonenvironmentalsignals,cellschoosetouseoneoranothersetofgenes,andtherebytoproceed along one or another developmental pathway. Whatmolecularmechanismsdeterminethesechoices?

The lifecycle isaparadigmfor thisproblem: theviruschooses one or another mode of growth depending upon

extracellular signals, andweunderstand inconsiderabledetailthe molecular interactions that mediate these processes. Webelievethatanalogousinteractionsarelikelytounderliemanydevelopmentalprocesses;byestablishingadescription for theparticularcaseof,wedevelopideasthatinformotherstudieseventhoughnoothercaselooksexactlylike.

The Introduction to this book describes some basic factsabout genes and how they work. It is designed to enable thereader with a modest knowledge of molecular biology tounderstandthefirstthreechapters.

These chapters then describe the process of sdevelopmentfromthreeperspectives:fromadistance,showingtheoverallpatternofeventsinvolvedintheinteractionbetweenthe virus and its host bacterium; more closely, describing incoarse molecular terms a key event in the process; and veryclosely, showing precise molecular interactions. The basicconceptsateachlevelarepresentedinthesechaptersinaseriesofpictureswithoutreferencetoexperiments.Atvariouspoints,our understanding of is related to developmental processesandgenecontrolinotherorganisms.

Chapter 4, more technical than the first three chapters,describes the principles of some of the key experiments. Theexperiments and the arguments based on them are easier tofollow if one knows the answers as outlined in the first threechapters.

The reader will see that we now have a coherentunderstanding of many aspects of gene regulation in . Ourintegrated picture accounts for the experimental observations,andmoreimportantly,predictsresultsofnewexperiments.Thisdegree of rigor is achieved, in part, because very few of our

models depend upon any isolated experimental observation;rather,theyarebasedonintegratedsetsofexperimentscarriedoutbothinthetesttubeandinlivingcells.

Thebooktakenasawholeisthusacasestudythatshowshowbiochemical andgenetic experiments construct a viewofpart of the world. I have avoided an historical approachadifferent and longer expositionwould be required to describehowourunderstandingdeveloped.

Oneofthecharmsofmolecularbiologyisthattheanswersit provides to fundamental questions for themost part can beeasily visualized. Simple pictures will usually do, and onlyrarelyneedweinvokeabstruseideas.Ourgoalistounderstandgene regulation in terms of the interaction of molecules. Aglimpse of the characteristic sizes and shapes of thesemoleculesoftenreveals,orhelpsusremember,howtheywork.Pleasetakethepicturesinthisbookseriously,butforwhattheyareasummaryofourcurrentviews.Ifullyexpectthatintheyears to come they will be redrawn as our understandingdeepens.

FortheirfriendlyadviceonthisprojectIwanttothank,inaddition to students and colleagues in my laboratory and atHarvard University, Alison Cowie, Nick Cozzarelli, NormDavidson, Hatch Echols, Gary Gussin, Gerhard Hochschild,WillMcClure,RussMaurer,HowardNash, JeffRoberts,BobSchleif, Hamm Smith, John Staples, Jim Watson, AdamWilkins, Keith Yamamoto, Michael Yarmolinsky, PatriciaZander,andespeciallySandyJohnson.BernardHirt suggestedtheideainthefirstplace.

Three people played especially important roles inproducingthisbookBenLewincoachedandeditedwithstyle

and understanding; Carol Morita created the illustrationsimaginatively and quickly; and Carol Nippert typed andretyped,organizedandreorganized,superbly.

MARKPTASHNECambridge,MA

January1986

INTRODUCTION

Some 40 years ago, Andr Lwoff and his colleagues at theInstitut Pasteur in Paris described a dramatic property of acertain strain of the common intestinal bacteriumEscherichiacoli.Ifirradiatedwithamoderatedoseofultravioletlightthesebacteria stop growing, and some 90 minutes later they lyse(burst), spewing a crop of viruses called into the culturemedium.The viruses are also called bacteriophagesbacteriaeatersor simply phages.The liberated phagesmultiply byinfecting freshbacteria.Many infectedbacteria soon lyse andproducenewphagebutsomesurviveandcarryinadormantform.Thesebacteriagrowanddividenormallyuntilthecultureis once again irradiatedthen eachof theseprogenybacteria,likethosewithwhichwestarted,lysesandyieldsanewcropofphages.Figure1.1showsapictureofthevirusanditshost.

Lwoff and his colleagues Franois Jacob and JacquesMonod realized that this switching between two states of thevirusfromthedormantforminthedividingbacteriumtotheactivated form in the irradiated bacteriumis a simpleexample of a fundamental biological process: the turning onandoffofgenes.

Genes determine the structures of molecules thatconstitute living cells. At any given time, each cellbe itbacterial or humanuses only a subset of its genes to directproductionofothermolecules,andwesaythattheseexpressedgenes areon and those not expressed areoff.We say in otherwordsthattheexpressionofthesegenesisregulated.

Asanexample,considerthedevelopmentofapersonfromafertilizedegg.Asthiscellandthenitsdescendantsdivideaprocess repeatedmillionsof timeseachnewcell receivesanidentical set of genes. Nevertheless some cells (for example,haircells)lookandactdifferentfromothers(forexample,skincells) because different genes are turned on in the differentcells.Theessentialpointisthatalthoughgenesaretransmittedunchanged (ignoring a few exceptions) from parental toprogeny cells, they may be expressed differently in variouscells.

Geneexpressionisregulatednotonlyduringdevelopmentbut also during the lifetime of the differentiated cell. Forexample, a skin cell changes colorwhen exposed to sunlight.The structure of the pigmentation gene does not change inresponse to the light; rather the extracellular signal, the light,turnsthegeneon.Totakeanotherexampleofgeneregulation:cancer cells multiply under conditions that their normalcounterpartsdonot,partlybecausecertaingenesareon(oroff)whentheyshouldnotbe.

Figure1.1. Electronmicrograph of and of anE. coli bacterium being infectedwith.Thediameterof the lambdahead is~600;magnificationsarex100,000(upper)andx8000(lower).PhotographskindlyprovidedbyRogerHendrix.

Biologistshavelongwishedtoknowhowgenesareturnedon and off normally during development and, aberrantly, in

diseased states. We are interested both in the molecularmechanisms of gene regulation and in how thesemechanismsare integratedinto interlockingcircuits thatensure theorderlyturning on and off of setsof genes. We wish to understandwhich steps are controlled by internal cellular programs andwhichbyextracellularsignals.

Returningtoouroriginalexamplephagewecannowappreciate the insightof thoseearlierworkerswhorecognizedinthegrowthof thisvirusinparticular itsability togrowintwodifferentmodesarevealingexampleoftheregulationofgene expression. Bacteria and their phages multiply quickly,and it is possible to combine genetics with biochemistry toanalyzegeneregulationmuchmoreefficientlythanispossiblewith cells of higher organisms. We turn now to a briefdescriptionofgenesandhowtheywork.

Our starting point is the structure of a gene, a piece ofDNA,byitselfaninertmolecule.ButDNAcarriesinformationinthesequenceofbasesalongitstwostrands.Thefourbasesadenine (A), thymine (T), guanine (G), cytosine (C)areattached to the two intertwined backbones so that A on onestrandisalwayspairedwithTontheother,andsimilarlyaGisalwayspairedwithaC.

Figure 1.2 shows three representations of a segment ofDNA.Inthefirstthesequenceof24basepairsiswrittenontwolines, the bases along the top line representing one strand ofDNAthe top strandand those along the bottom line theother strand. We say the sequences of the bases along thestrands are complementary because the base pairing rulesrequire that the sequence of either strand dictates that of theother.

Figure 1.2. Different ways to visualize DNA. The representation on the topemphasizes the complementary base pairing.The second shows the shape of thedouble helix with its major and minor grooves. The polarity of the strands isindicated by the arrow heads and tails. The two grooves are also seen in thebarber pole representation on the third line. (The precise number of base pairsperturnofthehelixis10.5andnot10.)

In the second representation in the figure, some 80 basepairsaremodeledinthetypicaldoublehelicalform.Notethatbecause one turn of the helix comprises about 10 base pairs,these 80 base pairs form about eight helical turns. Twoextendedgroovesofdifferentwidthsthemajorandtheminorgrooveswind around the surface of the helix. Thisrepresentation shows that thebackbones runalong theoutsideofthehelixwhilebasesfaceinwards.

Finally,inourthirdrepresentation,aDNAmoleculelooks

likeabarberpolebearingtracesofthetwostrandsofthehelix.Thisform,inwhichthevariousstructuralfeaturesofthehelixare simplified but kept in proportion, is used throughoutChapterOne.

The arrow heads and tails on each strand in the figureindicate another featureof theDNAdoublehelix: each strandhas a polarity or directionality, and the two strands run inoppositedirections.AsFigure1.3shows,thisstrandpolarityisaconsequenceof thefact that thechemical linkagesalongthebackboneofeachstrandareasymmetric.Forreasonsexplainedinthefigure,thearrowheadsandtailsarecalled,respectively,the3and5endsofthechain.

Figure1.3. Acloser lookatDNA.ThebackboneofeachDNAstrandcomprisesalternatingsugarsandphosphates.Directionalityisdefinedbythewayeachsugar

is attached to one phosphate by a 5 linkage, and to another phosphate by a 3linkage.Eachsugarisalsoattachedtoabasethatispairedwithitscomplementarypartner on the other strand.Three interactionshydrogen bondsform betweenG:Cbasepairs, and twobetweenA:Tpairs.Aguaninebase, attached toa sugar-phosphate,willaddtothe3endofoneofthestrands,andtwophosphategroupswillberemovedintheprocess.

Figure1.4. DNA replication. In the stemof thewishbone theDNA strands havenotyetcomeapart.Inthearmsthestrandsareseparatedandareusedastemplatesforformationofthecomplementarystrands.Asthestrandsarecopied,ApairswithT,andGpairswithC.

The principle of sequence complementarity explains howgenes are faithfully duplicated and, as we shall see in amoment,howtheyareexpressedaswell.Figure1.4showsthatas DNA replicates its two strands unwind, and each issequentially copied using the rules of base pairing. In thisprocesseachparentalstrandisusedasatemplateforformationof its complement, and every time a cell divides its twodaughtersreceivereplicasoftheparentalgenes.

A typicalgeneconsistsofasequenceofabout1000basepairs (100helical turns)withinamuch largerDNAmolecule.The first step in gene expression is always the same: thesequencealongoneofitsstrandsiscopied,ortranscribed,intoa linearmolecule calledRNA.As indicated inFigure1.5, thesequence of bases along theRNA is identicalwith that alongoneof theDNAstrandsand thecomplementof thatalong theother (the template). For our purposes it suffices to define ageneasonifitisbeingcopiedintoRNA,andoffifnot.

Some RNA molecules are end products that functiondirectlyinthecell.OtherscalledmessengerRNAs(mRNAs)specify the designs ofmolecules called proteins. There aremany kinds of proteins, including, for example, structuralcomponents,antibodies, andenzymes.The latter carryout theactualworkof the cell, including transcribingandduplicatingDNA.

Figure 1.5. Complementarity of mRNA with DNA. In RNA the base U is theequivalent of the baseT inDNA.Thus the sequence of thismRNA sequence iscomplementarytotheDNAsequenceofthebottomstrand.ThismRNA,aswellasthoseofthenexttwofigures,isgrowingintherightwarddirection.

Aproteinconsistsofastringofunitscalledaminoacids,whose sequence isdeterminedby the sequenceofbases alongthe gene. The mRNA is read (translated) sequentially,beginning at its 5 end. Each successive group of three basesspecifies one of 20 amino acids to be added to the growingproteinchain.Thefirstaminoacidoftheproteinchainiscalledtheproteinsaminoterminus,thelastitscarboxylterminus.

Each protein folds into a characteristic shape determinedby its amino acid sequence, typically forming an irregularglobulewhosesurfaceismarkedbycavitiesandprotuberances.HereandinChapterOnewewilldiscussexamplesofproteinspictured as blobs, and in Chapter Two we will see howcharacteristicsurfacefeaturesofagroupofproteinsinvolvedin

generegulationdeterminetheirfunction.Genes are transcribed into mRNA by an enzyme called

RNApolymerase.Theprocessbeginswith thebindingof thisenzymenearthebeginningofagenetoasitecalledapromoter,a region extendingover some60base pairs.Figure1.6 showsanRNApolymerasemoleculeabouttobindtoapromoterofatypicalbacterialgene.Wedonotknowmuchabouttheshapeofthisenzymebutweknowitsapproximatesize.

Following the initial binding, polymerase travels awayfromthepromoteralongthegene,synthesizingthemRNAasitmoves, as inFigure 1.7. Note that as polymerase moves itcontinuously unwinds and then rewinds successive shortregions of DNA. This transient unwinding separates the basepairs so that the sequencealongoneof the strandsbecomesatemplateforformationofthecomplementarymRNA.

Figure1.6.ThepromoterandRNApolymerase.RNApolymerase,whenboundtothepromoterat thebeginningof thegene,coverssix turnsof thehelix,about60base pairs.This simplified representation of polymerase ignores the fact that thislarge enzyme consists of several chains of different amino acid sequence. Thearrowshowsthedirectionoftranscription,whichbeginsatthebasepairjustabove

thearrowstail.

Figure1.7.Transcriptionofagene.AsthepolymerasemovesalongtheDNA,theunwoundDNAsegment,aboutonehelicalturninlength,isalwaysincontactwiththe same part of the polymerase.At any given instant the template base beingcopied lies in the unwound region and is about 20 base pairs frompolymerasesnose.WhenpolymerasereachesthestopsignalitfallsofftheDNAandreleasesthemRNA.

Eachpromoterpointspolymeraseineitheroneortheotherdirectionalong theDNAhelix,andas theenzymemoves inagiven direction it copies only one of the strands intomRNA.The polarity of themRNA chain is opposite that of theDNA

templatestrand.As represented inFigure 1.8, two polymerase molecules

moving in opposite directions copy different strands. ThepolymerasemovingleftwardcopiesthetopstrandasdefinedintherepresentationofDNAatthetopofFigure1.2.WhenweconsiderthevirusingreaterdetailweshallseethatitssinglelongDNAmoleculeconsistsofmanygenes,someofwhicharetranscribedrightward,someleftward.

RNApolymerasecanbehelpedorhinderedinitsattemptto transcribeagenebyregulatoryproteins thatbind tositeson the DNA called operators. A negative regulator preventstranscription, and a positive regulator increases (stimulates)transcription of a gene. In Chapter One we will explain theworkings of one regulatory proteinthe repressorthat isbothapositiveandnegativeregulatoroftranscription.

Wewilloftensaythataparticularregulatoryproteinbindsto a specific operator site (or sites) on aDNAmolecule.Wemeanby this that theprotein isusually tobe found there,butthat it can quite readily (and often does) fall off that site.WhetheranotheridenticalproteinquicklybindsagaindependsuponitsconcentrationanditsaffinityfortheDNAsite.

ADNAmoleculemayhavemorethanonesiteabletobinda particular protein. Such sites can vary in the strength withwhichtheybindtheprotein.Ifonesiteisweakerthananotherthen,atanygiveninstant,andatlowproteinconcentrations,thestrongersite ismoreapt tohaveaproteinboundto it.But,asillustrated inFigure 1.9, at high protein concentrations thedifference in affinitieswouldbe ignoredandboth siteswouldusually be filled. This state of dynamic equilibrium obtainsbecause the bonds involved in protein-DNA interactions are

muchweakerthanthose(forexample)thatholdthelinksoftheproteinchaintogether.

The first four chapters of this book are designed asfollows. ChapterOne describes how the regulatory proteins repressor and Cro bind to DNA and interact with RNApolymerase inamanner thatdetermineswhichpromoterswillbeusedtoinitiatetranscription.Thesecomponents,constitutinga genetic switch, control the remarkably efficient change ingene expression that is triggeredwhen a bacterium bearing adormantisirradiatedwithultravioletlight.

Figure 1.8. Divergent transcription. The sequence of the mRNA of gene B iscomplementarytooneoftheDNAstrands,andthatofgeneAiscomplementarytotheotherDNAstrand.

Figure1.9.Bindingofaregulatoryproteintoastrongandaweakoperatorsite.Aregulatoryprotein fillsonly the strongoperator site at lowconcentration,but fillsboth operator sites at high concentration. Put anotherway, the protein first binds

thestrongsite,thentheweaksite,asitsconcentrationincreases.

To understand the logic of the switchmechanism at thisstage,weneedtoinvokeknowledgeofmolecularstructure,butonly crudely.We pictureDNA as a rigid rod bearing sites towhichRNApolymeraseandtheregulatoryproteins,picturedasspheresordumbbells,bindtopromotersandoperators,turninggenesonandoff.Manysimplepicturesareusedtoportrayeachcomponent and each interaction separately so that themechanismsbecometransparent.

Chapter Two describes the structures of regulatoryproteinsinmuchgreaterdetailthanChapterOne.Itelucidatesasimpleandgeneralmechanism,withsomevariations,bywhichthese proteins recognize specific base sequences amongmillionsofbasepairs.Thestructureofa regulatoryprotein isshowntobecomplementarytopartoftheDNAstructure;ifthesequencesarecorrect, the twomolecules fit together like lockandkey.Analysis at this levelofdetail enablesus to surmisehowaregulatoryproteincanturnageneonoroff.

ChapterThreetracesthepatternsofgeneactivityasthephagelysesthecell,oralternatively,asitbecomesdormantinthecell.Thefirstfewstepsofgeneregulationthatoccuruponinfectionareidenticalforbothpathways.Atthecriticalstepthestateofthehostissensedbyaphageregulatoryproteinthatdetermines which pathway subsequent events shall take. Thisdecision is an instructive example of how the environmentcan influence gene regulation during development. Onceinitiated, the regulatory sequence along each pathway is acascadegroups of genes are turned on and off sequentially

accordingtoaninternallydeterminedprogram.ChapterFouroutlinessomeof theexperimentalbasesfor

manyof thepictures inChapterOneandChapterTwo. Ihavesimplified some of the arguments and have not tried to becomplete. Nevertheless the reader unfamiliar with thetechniques used in experimental molecular biology will findthischaptermoredifficult to follow.Thischapterneednotberead beginning to end, but rather might be dipped intoaccordingtothereadersspecialinterest.Thechapterconcludesbynotingsomeunsolvedproblems.

Themainpurposeofthisbookistoprovideanaccountofthemechanismsusedtoregulateindividualgenesandofhowthese mechanisms interconnect to form regulatory networks.From awider perspective,we believe that a central aspect ofthegeneralquestionofdevelopmenthowacomplexorganismdevelopsfromafertilizedegginvolveselaboratenetworksofdifferentiallyregulatedgenes.Atvariouspoints,particularlyattheendofChapterThree,Ihavethereforedrawnsomeparallelsbetweendevelopmentalprocessesandgeneregulationinandinhigherorganisms.

Anoteonnomenclature:genesaredenotedwithitalicizedletters, usually but not always in lowercase, for example,cro,recA,lexA,N,Q;andtheirproteinproductswithRomanletters,first letter capitalized, for example, Cro, RecA, LexA, N, Q.Sometimes for historical reasons, the protein has a specialname,forexample,repressor,encodedbythegenecl.

CHAPTERONE

THEMASTERELEMENTSOFCONTROL

Thegenesofphageconstitutea singleDNAmoleculeitschromosomewrappedinaproteincoat(Figure1.1).Thecoatis an elaborate structure with a head and a tail, togethercomposedof some15different proteins, all encodedby the chromosome.Thephageparticleisinfectious:itattachesbyitstailtothesurfaceofanE.colicell,drillsaholeinthecellwall,andsquirtsitschromosomeintothebacterium,leavingitscoatbehind.LambdaisanobligateparasiteitmustinjectitsDNAintothebacteriumtomultiply.

One of two fates awaits the -infected bacterium, asillustrated inFigure1.2. In somecells thephagechromosomeenters the lyticcycle:varioussetsofphagegenes turnonandoff according to a precisely regulated program, the chromosome is extensively replicated, new head and tailproteinsaresynthesized,newphageparticlesareformedwithinthe bacterium, and some 45 minutes following infection thebacteriumlysesandreleasesabout100progenyphage.

Inothercellstheinjectedphagechromosomelysogenizesitshost:allbutoneofthephagegenesareturnedoff,andonephage chromosomenow called prophagebecomes part ofthe host chromosome.As the lysogenthe bacterium bearingthe prophagegrows and divides, the prophage is passively

replicated and quiescently distributed to the progeny bacteria,allthewhileaspartofthehostchromosome.Thisprocessmayberepeated indefinitely,and ifunperturbed thesegrowinganddividinglysogenicbacteriaonlyveryrarelyproducephage.

When irradiatedwith ultraviolet light, however, virtuallyevery lysogen in the population will lyse and produce a newcropof.Theultravioletlightturnson,orinduces,previouslyinert phage genes and lytic growth ensues.Many agents, likeultraviolet light, induce lyticgrowth in lysogensbydamagingthehostDNA.Thephagechromosomeusesbacterialenzymesto sense the impendingdemiseof its hostaneffect of theinducingagentand,abandoning itspreviouslyusefulschemeofpassivereplication,itentersthelyticpathway.

Thischapterexplainshowthephagegenesaremaintainedstably in the lysogenic state and are then switched with highefficiencytoasecondstatelyticgrowthuponexposureofalysogen to an inducing signal. Viewed broadly, the switchworksasfollows.

Figure1.1.Aparticle.Thechromosomesome50,000basepairsofDNAiswrappedaroundaproteincoreinthehead.

In a lysogen the single phage gene that is expressedencodes the repressor. This protein is both a positive and anegative regulator of gene expression.By binding to just twooperatorsonDNAitturnsoffalltheotherphagegenesasitturnson its owngene. (How the repressor-encodinggenegetsturned on in the first placeimmediately following infectionwhen there is no repressor presentwill be explained inChapterThree.)

Althoughthereisonlyoneprophageinalysogenthereareabout 100 active molecules of repressor, and the excessrepressor isfreetobindtoanyadditionalchromosomesthat

mightbeinjectedintothecell.ThishastheresultillustratedinFigure1.3:cannotgrowlyticallyona-lysogen.Thelysogenissaidtobeimmunetoinfection.

Ultraviolet irradiation of lysogens inactivates repressor.As a result a second phage regulatory proteinCroissynthesized. Cro, which promotes and is required for lyticgrowth,alsobindsDNAinfactitbindstothesameoperatorsitesasdoesrepressor,butwithoppositephysiologicaleffects.These two regulatoryproteins, togetherwithRNApolymeraseand their promoter and operator sites onDNA, constitute theswitch.

Simplifying somewhat we say that the switch has twopositions:inthefirst(thatis,inalysogen)therepressorgeneison,butthegeneencodingCroisoff;andinthesecond(thatis,duringlyticgrowth)thecrogeneisonbuttherepressorgeneisoff.Wenowdescribe theapproximatesizesandshapesof theswitchcomponentsandhowsomeoftheminteract.

Figure 1.2. Growth of phage .The injected chromosome may either lyse orlysogenize the host. Ultraviolet irradiation of a lysogen induces lytic growth.Induction of lysogens was first demonstrated for a prophage of the bacteriumBacillusmegaterium.

Figure 1.3. Immunity of a -lysogen. Lambda phages inject their chromosomesinto a -lysogen, but repressor molecules (R) immediately turn off the genes ofthese superinfecting chromosomes, just as they turn off the genes of theprophage. Immunity is thus caused by the same repressor that maintains theprophageinitsdormantstate.

COMPONENTSOFTHESWITCH

DNA

ThegenesthatencoderepressorandCronamely,clandcroareadjacentonthechromosome.Thesegenesaretranscribedinoppositedirections,divergently,asshowninFigure1.4.Thestartpointsoftranscriptionofthesetwogenesareseparatedby80 base pairs, and in this region lie two kinds of sitespromoter and operatorto which protein components of theswitchcanbind.

Figure1.4showsthateachofourgenescl andcrohas itsownpromoter.Theclpromoter,calledPRM,pointspolymerase

leftward, and thecro promoter,PR, points polymeraserightward. The surfaces of the DNA molecule that comprisethese two promoters are shaded in the figure.Note that thesetwopromotersareadjacentbutdonotoverlap.

Three adjacent sitesOR1,OR2, andOR3compriseOR,therightoperatorof,alsoshowninFigure1.4.RepressorandCro bind to these sites to regulate the activities of the twopromoters.Notetheorderoftheoperatorsitesasshowninthefigure;eachsiteoverlapsoneor theotherpromoter,or, in thecaseofOR2,bothofthepromoters.

Eachofthethreeindividualoperatorsitesis17basepairs;their sequences, although similar, are not identical, and theregulatoryproteinscandistinguishbetweenthem.Forexample,considering any two operator sites, one might have a higheraffinity than theotherforCro.Thiswouldmeanthat,atsomespecifiedconcentrationoftheprotein,repeatedsnapshotsoftheoperator would reveal that a Cro molecule wasfound morefrequently at the stronger binding site. At higher proteinconcentrations, the snapshotswould revealCro usually boundtothesecondsitealso.

Figure1.4.AshortsegmentoftheDNAmolecule.Twoback-to-backpromoters(PRM andPR) send polymerase traveling in opposite directionsleftward totranscribe the repressor gene (cl) and rightward to transcribe thecro gene. Thetripartiterightoperator(OR)overlapsthetwopromoters.Eachofthethreepartsoftheoperatoriscalledanoperatorsite.

A note on nomenclature: the rationale behind thenomenclatureof the switchelementswill notbecomeentirelyclearuntil later,particularly inChapterThree.Briefly,PRandOR stand for therightpromoterandoperator.Therearealsoaleftpromoterandoperator(PLandOL)thepropertiesandrolesof which are considered in later chapters. The namecldistinguishes this gene from genescll andclll;cro stands forcontrol ofrepressor andothergenes,because that iswhatCrodoes.PRM stands forpromoter ofrepressormaintenance todistinguish it from a related promoter that we discuss inChapterThree.

RNAPolymerase

RNApolymerase,theenzymethattranscribesgenesfromDNAtoRNA, isprovidedby thebacterial host.WhenboundatPR,polymeraseispoisedtotranscribe(rightward)thecrogene,andwhen atPRM,polymerasecanmove leftward, to transcribe theclgene.

Figure1.5illustratesthepositionsthatwouldbeoccupiedif both promoters were occupied by polymerases at the sametime. In fact, these two promoters are never occupiedsimultaneously in the celldepending on the position of theswitch,polymerasecanbindtooneortotheother.Thus,asthefiguresuggests,inthepresenceofrepressorpolymerasemightbind toPRMbutnever toPR,andviceversainthepresenceofCro.OneoftheimportantquestionsofthischapterishowdorepressorandCrohavetheseoppositeeffects?

An important difference between the promotersPR andPRM is thatRNApolymerasebindsandbegins transcriptionattheformerwithouttheaidofanypositiveregulatoryprotein.Incontrast,polymeraseworksefficientlyatPRMonlyifhelpedbyanactivatorprotein,aroleplayedbytherepressor.

Figure1.5.Aneventthatneveroccurs.RNApolymerasemoleculescouldoccupybothPR andPRM on the sameDNAmolecule.But this does not happenin thepresence of repressor, polymerase may occupyPRM, but neverPR, and in thepresenceofCro,PRmaybeoccupied,butnotPRM.

TheRepressor

TherepressorshowninFigure1.6isaproteinof236aminoacids that folds into two nearly equalsized blobs, calleddomains,connectedbyastringof40aminoacids.Thedomainsare called amino and carboxylthe former includes the firstaminoacidofthechainandthelatterthelastaminoacidofthechain.

Two of the chains ofFigure 1.6, called monomers,associate to form dimers as shown inFigure 1.7. The dimerforms largely because of contacts between carboxyl domains,theaminodomainscontributingonlyslightlytothisreaction.Ina lysogenic cell about 95% of the repressor molecules areassociatedasdimersasindicatedinFigure1.8.

Figure1.6.Therepressor.Theaminodomainofrepressorcomprisesaminoacids1-92anditscarboxyldomainofresidues132-236.Theremaining40aminoacidsconnectthetwodomains.

Figure 1.7. Dimerization of repressor. Repressor monomers associate to formdimerswhich, in turn, dissociate tomonomers.We say that repressormonomersareinequilibriumwithdimers.Astheconcentrationincreases,alargerfractionoftherepressorispresentasdimer.

Figure1.8.Repressorinalysogen.Mostoftherepressorinthelysogeniccellisinthe dimer form. The single longE. coli chromosome contains one integratedprophage towhich repressors are bound tightly.The rest of the dimers associatelooselywithotherpartsofthebacterialchromosomeorfloatfreelyinthecell.

Figure1.9.Arepressordimerboundtoone17basepairoperatorsite.Eachaminodomain is centered on a segment of the major groove, a point we return to inChapterTwo.

RepressordimersusetheiraminodomainstobindtoDNAas shown inFigure1.9, and each of the three sites inOR canbindonerepressordimer.Therepressordimerbindsalongonefaceofthehelixateachsite.

Cro

Croshown inFigure1.10ismade of only 66 amino acids,foldedintoasingledomain.TheaffinityofCromonomersforeach other is high and virtually all Cro in the cell exists asdimers. In the absenceof repressor,Cro canbind to the threeoperator sites inOR. Figure 1.11 shows that one Cro dimerbindsalongthefaceofthehelixateachsiteandiscenteredinexactly the same way as the repressor dimer. These twoproteinsrepressor andCrobind to the same three operatorsitesbutplayopposingrolesintheswitchmechanism.

Figure1.10. LambdasCro.TheCromonomercontainsonly66aminoacids,butfolds into aglobular structure about themonomerdimer same size as repressorsaminodomain.

Figure1.11.OneCrodimerboundtoa17basepairoperatorsite.Crobindsalongthesamefaceofthehelixasdoesarepressordimer.

THEACTIONOFREPRESSORANDCRO

A key to understanding repressors action is to consider theeffectofattachingasinglerepressordimertoOR2,leavingOR1a n dOR3 free. This scenario is not observed under normalcircumstances, as we will explain, but it can be contrivedexperimentally, and the effects are revealing. The picture inFigure 1.12 shows that a repressor atOR2 performs the twofunctionsnecessaryformaintainingthelysogenicstate:itturnsoff thecro geneand it turns on the repressor gene.We nowconsider themechanismunderlyingeachof these functionsofrepressor.

NegativeControl

Repressor atOR2 turnsoff thecrogenebypreventingbindingofRNApolymerasetoPR.Itexertsthiseffectbycoveringpartof the DNA that polymerase must see to bind toPR. This

principleofexclusionunderliesmanycasesofnegativecontrol.

PositiveControl

Repressor atOR2 helps RNA polymerase bind and begintranscriptionatPRM,thepromotergoverningtranscriptionofclin a lysogen. The increase in transcription is roughly tenfold.Repressorwas namedbecause of its ability to turn off all theotherphagegenes.Onlylaterwasitrealizedthatrepressorisapositive regulator as well, increasing transcription of its owngene.

Figure 1.12. Repressor bound toOR2. In a lysogen repressor is rarely bound toOR2unlessitisalsoboundtoOR1.ButifitwereboundtoOR2only,itwouldturnonPRM and turnoffPR, socl butnotcrowouldbetranscribed.Justasaprotein-protein interaction holds two repressor monomers together in a dimer, so aninteraction between repressors amino domain and polymerase helps the enzymebindandbegintranscriptionatPRM.

Figure 1.13. Repressor bound atOR1. A repressor atOR1 only would preventpolymerase frombinding toPR.PRMwouldfunctiononlyata low(unstimulated)levelbecausethereisnorepressoratOR2tohelppolymerasebindthere.

The basis of repressors action as a positive regulator issuggested inFigure 1.12: the repressor atOR2 touches RNApolymerase atPRM. The repressor dimer bound toOR2increases the affinity ofPRM for polymerase becausepolymerase is held there not only by contacts with DNA butalsobyaprotein-proteincontactwithrepressor.

A repressordimerbound toOR2, in sum, repressesPRbyexcludingbindingofRNApolymerase to thatpromoter,but itencourages polymerase to begin transcription atPRM. Itprevents transcription to the rightbutaids transcription to theleft. (We will see in Chapter Two that this difference isaccountedforbythefactthatOR2isslightlyclosertoPR thantoPRM.)

We now consider the effects of a single repressor dimerbound either toOR1 or toOR3. AsFigure 1.13 shows, arepressoratOR1wouldblockbindingofpolymerasetoPR,but

would be too far away to affect polymerase atPRM. PRMfunctions only at a very low level, and is labeled off in thefigure, because no repressor is bound atOR2. AsFigure 1.14shows, repressoratOR3wouldblockbindingofpolymerasetoPRMandhavenoeffectonPR.

Figure 1.14. Repressor bound atOR3. A repressor atOR3 only would have noeffectonPR,whichwouldbeon.PRMwouldbeoff.

Figure1.15.Theoperatorinalysogen.InalysogenrepressordimersareboundprimarilytositesOR1andOR2,andoccasionallytositeOR3aswell.

A series of snapshots of the inside of a -lysogenwouldreveal the situation summarized inFigure 1.15. Repressor

dimersarevirtuallyalwaysboundatOR1 andOR2, butOR3 isusuallyfreeofrepressor.ThisarrangementofrepressorsatORturns offPRbut turnsonPRM:hencerepressor,butnotCro,issynthesized. How is this pattern of repressor binding in alysogendetermined?

CooperativityofRepressorBinding

Twofactorsdeterminetheinteractionofrepressordimerswiththethreeoperatorsites.Oneistheaffinityofthedimerforeachof the threesitesconsideredseparatelywesaythatrepressorhasanintrinsicaffinityforeachsite.Thesecondfactoristheinteraction between repressor dimers bound to adjacent sites.Just as repressor atOR2 helps polymerase bind to promoterPRM, so repressors can interact with each other to facilitatebinding.

Imagine a repressor dimer approaching a nakedoperator.Although this repressor might investigate all three sites, itwouldusuallyfixitselfasshowninFigure1.16 toOR1thisisa way of saying that of the three sites,OR1 has the highestaffinity for repressor. This binding immediately increases theaffinity ofOR2 for a second repressor dimer, because thesecond dimer not only contactsOR2, but it also touches thepreviously bound repressor. The result of this interactionbetween repressors atOR1 andOR2 is that these two sites fillvirtuallysimultaneously.

Figure 1.16 also shows that at higher repressor

concentrationOR3, as well asOR1 andOR2, are filled. Thisbinding of repressor toOR3 turns offPRM, as we havedescribed. SiteOR3 binds repressor more weakly than doesOR2, despite the fact that the intrinsic affinities of these twosites for repressor are about the same and are about tenfoldweakerthanthatofOR1.RepressorbindingatOR2isfacilitatedby the interaction with another repressor atOR1, but therepressoratOR3mustbindindependently.

WhydoesrepressoratOR2helppolymerasebind toPRM,butnothelpanotherrepressortobindtoOR3?Figure1.16,andin greater detailFigure 1.17, shows the answer. Repressor-repressor interaction involves contacts between the carboxyldomains of adjacent dimers. Once a repressor dimer at OR2interactswithanotherdimeratOR1,itisnolongeravailabletointeractwithadimeratOR3.

Figure1.16.RepressorbindingtothethreesitesinOR.OR1bindsrepressoraboutten timesmore tightly than doesOR2 orOR3, so repressor first binds toOR1.Asecond repressor very quickly binds toOR2, butOR3 continues to bind weakly,andisfilledonlyathigherrepressorconcentration.

We imagine that interaction between dimers atOR1 and

OR2 requires that the proteins lean toward one another, acontortion that isallowedbecauseof flexibilityof thepeptideconnecting the amino and carboxyl domains.A dimer atOR2leaningtotherightinFigure1.16,andcontactingadimeratOR1,cannot simultaneouslycontactadimeratOR3.ThereforeOR3mustfillindependently.RepressoratOR2caninteractwithpolymeraseatPRM,however,becausethatcontactismadewithrepressorsaminodomain,thedomainthatalsocontactsDNA.One of these amino domains is positioned just so thisinteractionoccurs.

If we have correctly described how repressor dimers atOR1andOR2interact,wemightexpectthatarepressoratOR2couldlean to the left and interactwith another atOR3 if norepressor were bound atOR1. Indeed, interaction betweendimersatOR2 andOR3occursinthespecialcircumstancethatOR1ismutatedordeletedsothatnorepressorcanbindthere.Inthat case, as shown inFigure 1.18, repressor dimers fill sitesOR2andOR3simultaneously.

The protein-protein interactionswe have been describingare examples of cooperativity. For example, we say thatrepressors bind cooperatively toOR1 andOR2. Becauserepressor dimers atOR1 andOR2 interact,or repressordimersatOR2 andOR3interact,wesaythecooperativityisalternatepairwise.

Figure1.17.Interactionbetweenadjacentrepressordimers.Thelinkerbetweentheamino and carboxyl domains of repressor is flexible, so a repressor atOR2 cancontactanotheratOR1.

Figure1.18. InteractionbetweenrepressordimersboundatOR2 andOR3. IfOR1ismutantsothatnorepressorbindsthere,repressoratOR2is free to interactwithanother atOR3.This interaction increases the repressor affinity ofOR2 andOR3aboutfivefoldabovetheirintrinsicaffinities.

INDUCTIONFLIPPINGTHESWITCH

In a lysogen, repressor bound atOR1 andOR2 keepscro offwhile it stimulates transcription of its own genecl, assummarized inFigure 1.19. Repressor is constantly beingsynthesizedasthecellsgrowanddivide,whereasthecrogeneremains silent. If the repressor concentration increasesasmight happen, for example, were cell division temporarilyinhibitedrepressor would bind also toOR3 to turn its owngene off.As the cell began dividing again and the repressorconcentration dropped to the proper level thecl gene wouldresume functioning,providingmorerepressor.Thusaconstantlevelofrepressorismaintainedinthecell,despitefluctuationsingrowthrate.

Figure1.19.RepressorandRNApolymeraseinalysogen.Inalysogen,repressorboundatOR1andOR2stimulatesPRMwhilesimultaneouslyturningoffPR.

In the cell, repressor continuously falls off the operator,onlytorebindortobereplacedbyanotherrepressormoleculethat happens to be nearby. The concentration of repressor is

highenough toensure that,atanygiven instant,OR1 andOR2areverylikelytobefilled.Thus,intheabsenceofaninducingagentthelysogenicstateisstablevirtuallyindefinitely.

Wearenowinapositiontofollowthedramaticeffectsofultraviolet light in a lysogen, beginning with the eventsillustrated inFigure1.20.Theprimary effect ofinducers suchas ultraviolet light and other inducing agents is to damageDNA.Inamannernotfullyunderstood,thisdamageleadstoaremarkable change in behavior of a bacterial protein calledRecA.

Figure1.20.RecAcleavageofrepressor.Repressoriscleavedbetweentheaminoacidsalanineandglycinelocatedinthelinkerbetweenrepressorsdomains.

Figure 1.21. Repressor cleavage and induction. Cleaved repressors cannotdimerize, and so, following irradiation,when repressors fall off the operator theyarenotreplaced.

Under normal circumstances, RecA catalyzesrecombination between DNA molecules, but when DNA isdamaged, thisprotein alsobecomesahighly specificproteasethat cleaves repressor monomers. (RecA also cleaves otherrepressors, thereby turning on genes that help non-lysogeniccellssurvivetheotherwiselethaleffectsofultravioletlight,asdescribedinChapterThree.)

Figure 1.20 shows that the cleavage occurs at a specificsitelocatedintheregionofrepressorconnectingtheaminoandcarboxyl domains. The separation of the amino from thecarboxyl domain effectively inactivates repressor, because theseparatedaminodomainscannotdimerize,andtheiraffinityforthe operator in the monomeric form is too low to result in

efficientbindingattheconcentrationsfoundinalysogen.Nowas repressor dimers fall off the operator there are too fewdimerstoreplacethem,asillustratedinFigure1.21.

Two changes result. First, as repressor vacatesOR1 andOR2therateofrepressorsynthesisdrops(becauserepressorisrequired to turnontranscriptionof itsowngene);andsecond,polymerasebindstoPRtobegintranscriptionofcro.

Cros action is less complex than that of repressor. AsFigure 1.22 shows, Cro dimers bind independently (non-cooperatively)tothethreesitesinOR.Unlikerepressor,Croisstrictly a negative regulator.Akey toCros action is that theorderofitsaffinityforthethreesitesinOR isoppositetothatof repressor.Figure 1.23 shows that the first Cro to besynthesized binds toOR3. This prevents polymerase frombinding toPRMandabolishesfurthersynthesisofrepressor.Atthispointtheswitchhasbeenthrownandlyticgrowthensues.

Figure1.22. Cro bound toOR.Crodimersbind independently toeachsite in thetripartiteoperator.

AsPRcontinues to functionandcro is transcribed,soaregenes to the right ofcro, whose products are needed for theearly stages of lytic growth (seeChapterThree).MoreCro is

made until it reaches a level at whichOR1 andOR2 are alsofilled and polymerase is prevented from binding toPR, asshown inFigure 1.24. Cro, therefore, first turns off repressorsynthesisandthen,slightlylater,turnsoff(ordown)expressionofitsownandotherearlylyticgenes.

Figure1.23. Order of binding of Cro dimers for sites inOR.The affinity of siteOR3 for Cro is about tenfold higher than that ofOR2 orOR1.After the first Crodimer has filledOR3, the seconddimer binds to eitherOR1 or toOR2.The orderwithwhichCrofillsthesitesisoppositetothatwithwhichrepressorfillsthesites.Thusonawild-typeOR,theaffinityforCroisOR3>OR1=OR2,whereasthatforrepressorisOR1>OR2=OR3.

Figure1.24.BindingCrotoOR3blockssynthesisofrepressorandbindingtoOR1andOR2turnsdownexpressionofitsowngene.

COOPERATIVITYSWITCHSTABILITYANDSENSITIVITY

We have described three forms of cooperativity involvingprotein-protein interactions that contribute to making theswitchmechanismhighlyefficient:Repressormonomersformdimers,theDNA-bindingformof

repressor.Thesedimersfreelydissociateintomonomers,andinthecellmonomersanddimersareinequilibrium.Onewaytodescribethebindingofadimeristosaythat

onemonomerhelpstheothertobindtheoperator,thatis,twomonomersbindcooperativelytoanoperatorsite;

Repressordimersbindcooperativelytoadjacentsitesintheoperator.ThepredominanteffectisthatarepressordimeratOR1helpsanotherbindtoOR2;

RepressoratOR2helpspolymerasebindandbegintranscriptionatPRM.The sum of these effects is best grasped by referring to

Figure 1.25 which plots, approximately, the activity ofPRversus the concentration of repressor. Note that in a lysogenenoughrepressor issynthesizedtorepressPRabout1000-fold.Over thefirst twofold or threefold drop in repressorconcentration from this high level, the activity ofPR remainsunchanged. In effect, repression is buffered against ordinaryfluctuations in repressor concentration, so that lysogens arerarely accidentally induced. But when the repressorconcentration has dropped about fivefold,PR respondsdramatically,functioningatabout50%ofitsfullyunrepressedlevel. This allows synthesis of Cro and of other lytic geneproducts,therebyflippingtheswitch.

Figure1.25. Repression as a function of repressor concentration in two systems.Byfollowingtheblacklinefromrighttoleftweseethatrepressionismaintainedina lysogenonlyuntil the repressorconcentrationdropsabout fivefold.Thesystemthen responds dramatically to any further drop in repressor concentration, andinductionoccurs.Incontrast,asingle-siteoperator-repressorinteraction(blueline)wouldreactmuchmoresluggishlytoachangeinrepressorconcentration.

Itisinstructivetoconsiderahypotheticalswitchdevoidofcooperativity. For example, ifOR contained only a single sitecorrespondingtoOR2,thesystemmightwork,butonlycrudely.If this site bound a repressor dimer tightly enough to ensure1000-fold repression ina lysogen, inductionwouldoccuronlyveryinefficiently.Over99%oftherepressorwouldhavetobeinactivated to trigger lytic growth, and this is a difficultrequirementtomeet.

Thepropertiesof repressor thusprovideananswer toaproblem of concern to developmental biologists: how arelatively mild change in the concentration of a regulatory

protein can reliably cause a switch in gene expression.Cooperativitybetweenrepressormonomersthatis,bindingofa dimer to each siteand between repressor dimers on theDNAmagnifiesgreatlytheeffectofadropinconcentrationofrepressor monomers. Moreover, the fact that repressorstimulates transcription of its own gene, acting as a positiveregulator,meansthatsynthesisofnewrepressormustdecreaseas the repressor concentration falls. When the repressorconcentrationdropstothecriticalpoint,lyticgrowthensues.

The two curves ofFigure 1.25, one describing thecooperative repressor system, the other a single-sitenoncooperative system, are similar to the curves thatdescribethebehavioroftheoxygen-carryingmoleculeshemoglobinandmyoglobin.Hemoglobin carries oxygen from lungs to tissues,andmyoglobinhelpsmoveoxygenwithinmuscle.Theoxygenpressure in tissues is only about fivefold lowerthan that inlungs; nevertheless, hemoglobin efficiently accepts oxygen inthelungsandreleasesitinthetissues.Thecurvedescribingthebinding of oxygen to hemoglobin resembles the highlycooperative black curve ofFigure 1.25. The four sub-units ofhemoglobin, each of which binds one oxygen molecule, bindoxygencooperatively,andthisbindingishighlysensitivetotheoxygenconcentration.Anoncooperativecurve,similarinshapeto that drawn in red inFigure 1.25, describes the binding ofoxygentomyoglobin.Eachmyoglobinmoleculebindsonlyoneoxygenmolecule,andthisbindingismuchlesssensitivetotheoxygenconcentration.

THEEFFECTOFAUTOREGULATION

Wehavenotedthat,inadditiontoactivatingtranscriptionofitsown gene in lysogens (by binding toOR1 andOR2) repressoru s e sOR3 to limit its own concentration. Through thisinteraction atOR3, repressor ensures that its concentrationnever exceeds the level at which the prophage can respondefficiently to the inducing signal. Too much repressor wouldinhibitinductionintwoways.RecAcleavesrepressormonomersonlyratherslowly;ifthe

repressorconcentrationweretoohigh,thefractionofrepressorcleavedwouldnotbesufficienttocauseinduction.

Evenifrepressorathighconcentrationswerecleaved,repressionwouldnotbelifted.Thereasonisthateventhoughcleavedrepressormonomerscannotformdimers,theseveredaminodomainscanstillbindtotheoperatornon-cooperatively.Athighenoughconcentrations,theseaminodomainswillfillthethreebindingsiteswhetherornottheyarepartofrepressordimers.Negative and positive self-control circuitsas

exemplifiedbythetwoaspectsofrepressorsself-regulationrespond quite differently to perturbations.Negative control isself-correcting, or homeostatic, whereas positive control isdestabilizing. If only negative control were operative, shouldthe repressor concentration increase or decrease, the rate ofrepressor synthesis would decrease or increase so as to bringthe concentration back to the equilibrium level. In contrast, apositive control circuit would respond by exacerbating anychange in repressor levelan increasing repressorconcentrationwouldincreasetherateofrepressorsynthesisand

afallingconcentrationwoulddecreasethatrate.

OTHERCASES

We know of many examples of phages other than that, inaddition togrowing lytically, form inducible lysogensof theirbacterial hosts.Do the switches in these cases resemble the switch? Consider two other phages434, which grows onE.coli, and P22, which grows onSalmonella typhimurium. 434-and P22-lysogens, like -lysogens, are efficiently induced byultraviolet light. Both phages encode a repressor and a Cro,whichactonaregionoftheirownDNAanalogoustosOR.

Despite differences in detail, the following descriptionholds for all three phages: the right operator contains threerepressorbinding sites, twoofwhich,OR1 andOR2, are filledby repressor in a lysogen. This state depends on interactionsbetween adjacent repressor dimers.Repressormonomers havetwostructuraldomainsandareinequilibriumwithdimers,theDNA-bindingform.RepressordimersoccupyingsitesOR1andOR2 turn off transcription fromPR and turn on transcriptionfromPRM.Uponultraviolet induction, therepressor iscleavedand thereby inactivated,and thefirstproteinsynthesizedfromPRisCro.CrobindsfirsttoOR3toturnoffrepressorsynthesisand later binds toOR1 andOR2 to decrease early lytictranscription. The fact that these features are widespreadsupportstheviewthattheyarefundamentalcomponentsoftheswitch.

FURTHERREADING:RELATEDREVIEWS

1.Gussin,G.,Johnson,A.,Pabo,C.,andSauer,R.(1983).RepressorandCroprotein:structure,function,androleinlysogenization.InLambdaII,R.W.Hendrix,J.W.Roberts,F.W.Stahl,andR.Weisberg,eds.)NewYork:ColdSpringHarbor),pp.93-123.

2.Johnson,A.D.,Poteete,A.R.,Lauer,G.,Sauer,R.T.,Ackers,G.K.,andPtashne,M.(1981).repressorandcro-componentsofanefficientmolecularswitch.Nature294,217-233.

3.Lwoff,A.(1953).Lysogeny.Bacteriol.Rev.17,269.4.Ptashne,M.(1984).Repressors.TrendsBiochem.Sci.9,142-

145.5.Ptashne,M.,Backman,K.,Humayun,M.Z.,Jeffrey,A.,

Maurer,R.,Meyer,B.,andSauer,R.T.(1976).Autoregulationandfunctionofarepressorinbacteriophage.Science194,156-161.

6.Ptashne,M.andGilbert,W.(1970).Geneticrepressors.Sci.Am.222,36-44.

7.Ptashne,M.,Jeffrey,A.,Johnson,A.D.,Maurer,R.,Meyer,B.J.,Pabo,C.O.,Roberts,T.M.,andSauer,R.T.(1980).HowtherepressorandCrowork.Cell19,1-11.

8.Roberts,J.andDevoret,R.(1983).Lysogenicinduction.InLambdaIIR.W.Hendrix,J.W.Roberts,F.W.Stahl,andR.Weisberg,eds.(NewYork:ColdSpringHarbor),pp.123-145.

CHAPTERTWO

PROTEIN-DNAINTERACTIONSANDGENECONTROL

Regulatory proteinss repressor and Cro, for examplebind tightly tospecificDNAsequences15-20basepairs long.Eachoftheseproteinsmustselectitsoperatorsitefromamongthe fivemillionor sobasepairsofDNA inabacterium.Thischapterexamines thestructuresof theseproteins toshowhowtheyaccomplishthisfeat.Theprinciplethatemergesissimple:the structure of the protein is complementary to the DNAstructureiftheDNAsequenceiscorrecttheproteinandDNAmolecules fit together like lockandkey.Wealsoshowhowaregulatory protein, once bound, can control gene expressionnegativelyorpositively.

THEOPERATOR

ThedrawingofFigure2.1revealshow,inprinciple,asequenceof double-helical DNA can be recognized. Edges of the basepairsareexposedinthemajorandminorgroovesthatrunalongthe helix. Each base pair (A:T, T:A, G:C, C:G) exposes adifferent pattern of chemical groups that a protein might

recognize. These groups are not the ones involved incomplementarybasepairingthosegroupsbecomeaccessibleonly if the strands separate, as happens during replication ortransiently during transcription. We will see that repressorandCrobearprotuberances thatpenetrate themajorgroove toreadtheDNAbasesequence.

EachoftheoperatorsitesrecognizedbyrepressorandCrois nearlybut not exactlysymmetric. To understand this,considertheperfectlysymmetricsequenceofFigure2.2.IfthissegmentofDNAwererotated180intheplaneofthepageandaround the dot at its center, the identical molecule would begenerated.Thedistinctivefeatureofthisparticularsegmentofdouble-stranded DNA is that its base sequence remainsunchangedbytherotation.

Figure2.1.AsegmentofDNA.PeeringintothemajorandminorgroovesofDNAweseethateachbasepaircanbeidentifiedbycharacteristicchemicalgroupsthatlie along the edges of the base pair.TheDNA need not be unwound to read its

sequence.

AnotherwaytodescribethesymmetryofthesequenceofFigure2.2istoimagineatinydemonstandingatthemiddleofthemolecule, theposition representedby thedot.Thedemon,facing right and then left, would see identical corridors ofchemical groups. We say that our sequence is twofoldrotationally symmetric.Wereonebasepair tobe changed thesequencewouldbenearly,butnotexactly,symmetric.

ThesequencesrecognizedbyrepressorandCroarelistedinTable2.1.Threeofthese17basepairsitesarefromsrightoperator,OR,andthreefromtheleftoperator,OL.Thesixsitesdiffersomewhatinsequence,andtheydonotallhaveidenticalaffinitiesforrepressorandCro.

Noneoftheoperatorsitesisperfectlysymmetric,andifweweretoexamineonlyoneofthemOR2,forexamplethesymmetry would not be particularly striking. But if we spliteach operator into two half-sites, and align the half-sites asshowninTable2.2,aclearpatternemerges.Thetableliststhefrequency with which various bases are represented at eachposition. The consensus half-site, written in double-strandedform,is:

TATCACCGCATAGTGGC

Figure2.2.AsymmetricDNAsequence.Thesequenceofthetopstrand,readleftto right, is the same as that of the bottom strand read right to left. The blackdiamondindicatestheaxisofsymmetry.

Table2.1.ThesixoperatorsitesrecognizedbyrepressorandCro.Thesitesare listed in theorderof their intrinsicaffinities fora repressordimer.Thecentralbasepair,theaxisofsymmetry,isshowninblue.

Table2.2.The12half-sitesoftheoperators.Each of the six full sites listed inTable 2.1 is represented here as two half-sites.Onlyone strand iswritten foreachhalf-site.Theupper strandcorresponds to theleft half-site of the top strand ofTable 2.1 reading left to right; the lower strandcorrespondstothebottomstrandoftherighthalf-siteofTable2.1,readingrighttoleft. The positions in the operator are numbered at the top. At the bottom thefrequency with which each base appears is given for each position. One of twobasescouldbechosentorepresentposition9foreachcase.

Each operator site is more or less closely related to thefollowingsymmetricsequence.Itcontainstwoconsensushalf-sitesandanaxisofsymmetrythatrunsthroughthecentralbasepair:

TATCACCGCCGGTGATAATAGTGGCCGCCACTATT

The sequence of bases at each operator site must provide apattern of functional groups recognized by repressor andCro.Eachof theproteinsmust alsobe able to distinguishbetweenthesites,bindingwiththeproperaffinityorderasdiscussedinChapterOne.WeexaminenextthestructuresonrepressorandCrothatrecognizethesesequences.

REPRESSOR

In the Introduction we noted that proteins fold intocharacteristicshapesdeterminedbytheiraminoacidsequences.Although different amino acid sequences usually fold intodifferent overall shapes, certain smaller structural motifs arefoundinmanyproteins.Onecommonmotifisthe-helix.

As shown inFigure 2.3, the -helix is formed by thespiraling of a single chain of amino acids. An importantdifferencebetweenthe-helixandDNAsdoublehelixisthat,in theprotein structure, thebackbone is on the inside and thecharacteristic groups of each residuethe amino acid sidechainsareontheoutside.

Figure2.3. A chain of amino acids unfolded and in the formof an -helix.ThesidechainsR1,R2,etc.aredifferentforeachofthe20differentaminoacids.Inthe-helix, these side chains protrude from the backbone which is shown hereenclosedinabarrel.Oneturnofthe-helixcomprises3.6aminoacidresidues.

Figure2.4.An-helixinamajorgroove.Thesidechainsthatprotrudefromthe-helix,notshownhere,wouldextendtotheextremitiesoftheDNAmajorgroove.

Figure2.4showshowan-helixfitsneatlyintothemajorgrooveofDNA.Thefunctionalgroupsalongasurfaceofthe-helixarepositioned to interactwithtouchchemicalgroupsontheedgesoftheDNAbasepairs.

Tounderstandhowrepressorusesan-helixtorecognizeits operator we must consider the structure of repressor ingreaterdetailthanourdumbbellrepresentationinChapterOne.As shown inFigure 2.5, repressors amino domain is foldedinto five successive stretches of -helix. Alpha-helix 3 liesexposedalong thesurfaceof themoleculethis is repressorsrecognitionhelix.

In the repressor dimer, the recognition helicesone oneach monomerare separated by the same distance thatseparates successive segments of themajor groove along oneface of theDNA.Figure2.6shows thatwhen thedimerdockswith DNA, each recognition helix fits into themajor groove.Thus the symmetry of the protein matches that of the DNA

whenthedimerispositionedontheoperator.

Figure2.5. Lambdarepressor.Thefive-helices thatcompriserepressorsaminodomain are connected by segments of the amino acid chain. Helix 1 is near theveryaminoterminusoftheprotein.Thestructuresofthelinkerandofthecarboxyldomain(C)arenotknown.

Figure2.6.Lambdarepressorboundtoanoperatorsite.Apairofrepressoraminodomainsfitsona17basepairoperatorsite.

Figure2.7. Bihelical units on theoperator.Twobihelical units are symmetrically

positioned on an operator site. Bihelical units are also called helix-turn-helixmotifs.

Figure2.8.Thearmsofrepressor.Sevenaminoacidsextendfromtheendof-helix 1. Bywrapping around theDNA they contact bases near the center of theoperator on its backside. Note that the DNA has been turned around, comparedwiththepreviousfigure,toillustrateitsbackside.

A second -helixhelix 2is highlighted in addition tothe recognition helix in Figures2.5 and2.6.Figure 2.7 showsthatthis-helixliesacrossbutnotinthemajorgroove.Helix2helps topositionhelix3 in themajorgrooveof theDNA.Werefer to these two-helicesasabihelicalunitorstructure.Asweshallsee,manyregulatoryproteinsuseasimilarpairof-helicesinbindingtoDNA.

Nowwesee, inprinciple,howrepressorbindsselectivelytoitsoperator.Onlyifthereisamatchbetweentheaminoacidsidechainsalongrepressorsrecognitionhelixits-helix3andDNA functional groups exposed in themajor groovewilltheproteinbindtightly.Tounderstandtheroleofsymmetryinthe binding, recall our symmetry-detecting demon. Were thedemontowalkalonghelix3ineithermonomer,fromaminotocarboxyl endthat is, toward helix 4itwould see the same

(or nearly the same) succession of protein-DNA interactions.(The reasonwe say nearly the same is that the sequence ofeachoperatorsiteisnotperfectlysymmetric.)

InadditiontopenetratingtheDNAwithitsrecognition-helix, repressor embraces the DNA with a pair of flexiblearmsthatextendfromitsaminoend.Astherepressorbindstheoperator, its armswrap around andmake specific contacts onthebacksideoftheDNAinthemajorgroove.Manyregulatoryproteins use recognition -helices to recognize specific basesequences,butthearmsofrepressorshowninFigure2.8maybeanexampleofalesswidelyusedsequence-readingdevice.

CRO

Figure 2.9 shows Cros structure. It includes three -helicesand, in addition, three regions that form so-called -sheets.(The -sheet is a second common structuralmotif found inmany proteins. Our pictures represent -sheets as flattenedarrowstodistinguishthemfromthe-helicalbarrels.)

Figure2.9.Cro.Thethreeflattenedarrowsrepresentthesegmentsofa-sheetthat,inadditiontoitsthree-helices,formCrosstructure.

Cros helix 3just as with repressoris its recognitionhelix.TherelativespatialorientationofCros-helices2and3is virtually identical with that of the corresponding pair ofhelices in repressor. It is remarkable to find two -helicespositioned so similarly to form a bihelical unit in differentproteins. In the Cro dimer, the symmetrically relatedrecognitionhelicesoneoneachmonomerfitintosuccessivesegmentsofthemajorgrooveasshowninFigure2.10.

Figure2.10.Croboundtoanoperatorsite.ACrodimerdockswithDNAinmuchthesamewayasdoesarepressordimer.

AMINOACID-BASEPAIRINTERACTIONS

The amino acid sequence comprising the recognition helix ofrepressor andCro are for themost part different as shown inFigure 2.11. This is not surprising. In each case, one surface(theinside)of thehelix fitsagainst thebodyof theprotein,and theoutsidesurface fits into theDNAsmajorgroove tomake specific contacts.The inside surfaces of the recognitionhelices differ because the bodies of the proteins with whichtheyinteractaredifferent inrepressorandCro.Thesequencesalongtheoutsidesoftherecognitionhelicesaresimilarbutnotidenticalalthough repressor and Cro bind to the sameoperatorsites,theydosowithdifferentrelativeaffinities.

Figure2.11alsoshowsthepatternofinteractionsbetweenaminoacidsintherecognitionhelicesofrepressorandCrowithbases in two operator half-sites. The pattern suggests howrepressor and Cro recognize the same operator sites, but

distinguishbetweenthem,repressorpreferringOR1 toOR3andviceversaforCro.

Both recognition helices begin with the sequenceglutamine-serine(Gln-Ser)andthendivergeinsequence.BothproteinsuseGlnatposition1andSeratposition2 tocontactpositions 2 and 4, respectively, of the operator. These twopositions in theoperatorare just those,asshowninTable2.2,whose identities are invariant in all the operator half-sites.Thus, repressor and Cro use identical amino acids to contactbasesthatareidenticalinalltheoperatorsites.

Figure 2.11A. Pattern of amino acid-base pair interactions. To see how theindividual units of these recognitionhelices are oriented andnumbered, comparewithFigure2.3andFigure2.11B.Ineachoftheproteinsthesidechains(Rgroups)ofaminoacids1,2,5,and6pointtowardtheDNA,andthoseofresidues4and7pointtowardthebodyoftheprotein.ThearrowconnectingtheAlarepressortotheT:A base pair at position 5 is dashed because, although the presence of theAlacauses a preference for thatT:A, the basis for this preference is not known.Thefigureomitsthreeadditionalinteractions:aLysresidueinrepressorsarmandanAsnresiduejustbeyondtheendofrepressorshelix3bothcontacttheC:Gfoundat position 6 of bothOR1 andOR3.And, at position8, repressors arm,which isnot shown,prefersG:C toT:A.Onlyonehalf-site is shown for eachoperator. Inbothcases,theotherhalf-siteistheconsensussequenceofTable2.2.

Figure2.11B.TherecognitionhelicesareunfoldedfromtheirpositioninthemajorgrooveofDNA.Notethecorrespondencebetweenthepositionsoftheprotrudingamino acid side chains and the contacted bases. The bases in the operator arenumberedasinTable2.2 ,andthe threebasepairs thatdistinguishOR1 fromOR3arestarred.Thediamonddenotesthecenterofsymmetryoftheoperator.

AminoacidsotherthantheconservedGln-Serpairenablethetwoproteinstodistinguishbetweentheindividualoperatorsites.Thus,forexample,asparagine(Asn)inCrosrecognitionhelix contacts position 3 in the operator, preferring the basepair found in thenon-consensushalf-siteofOR3 to that foundin the corresponding position inOR1.Thearmof repressor,

which contacts positions 6 and 8 in the operator, also helpsrepressortodistinguishbetweenthesites.

Figure 2.12 shows in detail one of the amino acid-baseinteractions indicated inFigure 2.11. Thus, Gln makes twobondstopositionsonthebaseadenine(A)exposedinthemajorgroove.

Figure2.12. Anaminoacid-basepaircontact.Glutamine isshowncontacting thebaseA in the major groove.Two hydrogen bonds are formed between the sidechainoftheaminoacidandtheedgeofthebase.

Figure2.13shows the identitiesofcertainaminoacidsatcorresponding positions of the bihelical structure that are thesameorarechemicallysimilar(con-served)inrepressorandCro. (These conserved amino acids are not those involved inrecognitionofspecificsequences.)Threeof theseaminoacids

areintheelbowbetweenthetwohelices,andtwo,connectedbya line in the figure, lie one in each helix. These residuesevidentlyservetomaintaintheconstantanglebetweenthetwohelices.A sixth conserved residue is found at the top of thehelixprecedingtherecognitionhelix.Thisaminoacidinteractswith a phosphate in the backbone of one of theDNA strands,thereby helping to position the bihelical structure properly ontheDNA.

Figure 2.13. Conserved positions in the bihelical units of repressor and Cro.RepressorandCroeachbearanaminoacidinhelix2thatinteractswithanotherinhelix3alanineandvalineinthecaseofrepressor,andalanineandisoleucineinthe case of Cro. These bonds help position the two helices, as do the circledresiduesintheelbows.Thesidechainofmethionine(Met)ischemicallysimilartothoseofleucine(Leu)andvaline(Val).

Figure2.14. Phosphates in contactwith repressor bound atOR1.Thephosphatescontactedby repressor lie alongone faceof thehelix and are symmetric about atwofold axis through the center of the operator. Cro contacts a subset of thesephosphates.

Manyknownandputative specificDNA-bindingproteins-from an array of organismshave a pattern of conservedresiduesidenticalorcloselyrelatedtothatfoundinrepressorandCro. In several cases other than repressor andCro, thispatternofaminoacidshasproveddiagnosticofabihelicalunit,one member of which is a recognition helix. The relevanthomologies do not extend over the entire bihelical regions;rathertheyareconfinedtothosefewregionsthatevidentlyfix the spatial relation between the members of the bihelicalunit.

Lambda repressor and Cro bind very tightly to theiroperators, amatter towhichwe return inChapterFour and inAppendix1.Inourdiscussionthusfarwehaveemphasizedthedeterminants of specificity, namely, specific interactionsbetween DNA functional groups and the amino acids in therecognition-helixand,inthecaseofrepressor,theendofitsflexible arm. But some of the tightness of binding, probably

most, comes from interactions between other parts of theproteinandtheDNA.Forexample,whenrepressordockswithitsoperatortheproteininteractswithphosphatesthatliealongthe backbones of theDNAhelix, shown inFigure2.14. Theseandother tight interactions are allowedonly if the specificityprobesfindtheproperDNAgroupswithwhichtointeract.

We are just beginning to decipher the interactions ofaminoacidchainswithbasepairs that allowspecificbinding.We suspect that in general nomore than three or four aminoacidsonanyparticularproteinmonomerdeterminespecificity.DoestherecognitionhelixpresentitselfidenticallytotheDNAineverycasesothataminoacidsatspecifiedpositionsinteractwith bases at specified positions in the operator? Is there asimplecodedescribingaminoacid-basepairinteractions?

We have treated DNA as a rigid rod but this is asimplification. We know, for example, that the preciseparametersdescribingthehelicalDNAstructurearedeterminedinpartbysequence.Willthesesequence-specificalterationsinstructureinfluenceprotein-DNAinteractionssignificantly?Itislikely, for example, thatbases not actually contacted by abound protein might nevertheless influence the binding bysubtlyalteringlocalDNAstructureorflexibility.Wedonotyetknowhowimportantsuchfactorsare.

THEPROMOTER

The enzyme RNA polymerase inE. coli recognizes manypromoters near the beginning ofmany different genes.RecallfromtheIntroductionthatapromoterspansabout60basepairs,

including20basepairsdownstream(thatis, inthedirectionitwilltravel)fromwheretranscriptionbegins.Ingeneral,notwopromoter sequences are identical, but all have twocharacteristic blocks of sequence: one centered about 10 basepairsupstreamoftheRNAstartsiteandtheotherabout35basepairsupstream.Byconvention, thefirstDNAbasecopied, theRNA start site, is numbered +1, and so these conservedpromotersequencesarecenteredatpositions-10and-35.

BycomparingmanysequenceswecandeduceaconsensuspromoterwiththetwoblocksofsequenceshowninFigure2.15.Anygivenpromoterhasasequenceat thesepositionsmoreorlessclearlyrelatedtotheseconservedelements.

The sites of the promoter contacted by polymerase liealong one face of the helix from about position -10 to -40.Between -10 and +1 the DNA is opened so that one of thestrands can be copied into mRNA. The structural features ofpolymerase that enable it to bind selectively to promoters arenotknown.

In general a promoter that has a good match to theconsensussequence in the-35and-10regionswillworkwell.Butifthesequencedeviatesgreatlyfromtheconsensus,thenanactivatorproteinisusuallyrequiredtohelpthepolymerasebindandbegintranscriptionefficiently.

Table2.3comparestheconsensuspromotersequencewiththatactually foundatPR andPRM.PRmakesabettermatchatboththe-35regionandthe-10regionthandoesPRM.ThismaybewhyRNApolymerase can bind efficiently toPR andbegintranscription without the aid of any regulatory protein, butrequires anauxiliaryprotein repressorboundatOR2if it

istobindefficientlyandbegintranscriptionatPRM.

Figure2.15.Aconsensuspromoter.Thispromoterdirectstranscriptionrightward.The first base copied is at position +1, and -10 and -35 identify bases at thosepositionsupstreamofthetranscriptionstart.Theconservedsequencearound-10is sometimes called the TATA box. The separation between the conservedelements (TATA and -35) varies in different promoters between 15 and 18 basepairs,with17basepairsbeingoptimal.

Table 2.3. Lambda promoter sequences compared with the consensus promoter.Thedifferencesareshowninblue.

GENECONTROL

Wearenowinapositiontounderstandhowthepromoterand operator sequences are arranged so that the effects ofrepressorandCro,describedinChapterOne,arerealized.

Figure2.16showstheDNAsequencethatextendsfromclt ocro. Compare withFigure1.4, andnoteherehowpromotersequences overlap (interdigitate with) operator sequences.Aswe saw inChapterOne (seeFigure 1.13) whenOR1 is filled

withrepressororwithCro,RNApolymeraseisexcludedfromPR because either bound protein covers part of the surface oftheDNAhelix thatmustbeoccupiedbyRNApolymerase.AtOR3thesameruleholds:eitherregulatoryproteinwouldblockbinding of RNA polymerase toPRM. AtOR2 the situation ismoredelicate.

Recall from the previouschapter thatOR2mediates bothnegativeandpositivecontrol:repressorboundatthissiteturnsoffPR, while it stimulatesPRM. The mechanism of negativecontrolisthefamiliarone:repressorboundtoOR2wouldcoverpartof theDNAsurface thatpolymerasemust see tobindPR.Thepromotersurfaceoverlappedbyrepressoratthissiteislessthan ifOR1 were occupied, but even this degree of overlapsufficestoeffectrepression.

Figure2.16. Linear relationship between promoter and operator sites aroundOR.Some base pairs serve dual functions in the region betweencl andcro. Forexample,threeofthebasepairsofOR2formpartofthe-35regionofPR.

Figure 2.17. Lambda repressor as an activator of transcription. Repressor aminoacids that interact with polymerase to mediate positive control are located alonghelix2andinthebendbetweenhelix2andhelix3.

Howdoes repressor bound toOR2 stimulatePRM? Figure2.16 shows thatOR2 is one base pair closer toPR (countingfrom the transcription start site) than it is toPRM.Becauseofthisdifference,repressoratOR2doesnotcoveranypartofthesurface ofPRM.Rather, repressor atOR2closelyapproachestouchesRNApolymeraseboundatPRM.Asnoted inChapterOne (seeFigure 1.12), this interaction helps polymerase bindandbegintranscriptionatPRM.

The surface of repressor that touches polymerase andmediatespositivecontrolisindicatedinFigure2.17.Theamino

acidson thissurfacepatcharespeciallyconfigured to interactwith polymerase and, if they are changed, the repressor canbind toOR2butcannolongerstimulatePRM.WeimaginethatwereOR2 positioned one base pair closer toPRMtherebymimicking its relation toPRrepressor atOR2 would blockbindingofpolymerasetoPRMratherthanhelpit.

Cro atOR2 also blocks polymerase from binding toPR.The region ofPR covered by Cro atOR2 is the same as thatcoveredby repressor atOR2. IfCro isboundonly toOR2anartificial situation that can be contrivedit fails to stimulatePRM. The reason is that, although Cro might be positionedproperlytocontactpolymeraseatPRM,itlackstheappropriateaminoacidsalong thecontactingsurface thatwouldfavorablyinteractwithpolymerase.

It is worth emphasizing that contemplation of a linearrepresentation of operator and promoter sequences can bemisleading.Forexample,Figure2.16seemstosuggestthatOR2overlaps bothPR andPRM such that repressor bound therewould repress bothpromoters.The three-dimensional analysismakes clear the fact that proteins can recognize overlappingsequencesbutbeboundondifferentsurfacesoftheDNAhelix.

The analysis of this chapter suggests that a protein willrepressapromoter if itcoverssomesurfaceof theDNAhelixto which polymerase must bind. There are various ways thatoperator and promoter sequences can be arranged to ensurerepression, but in each case, the sequencesmust overlap (thatis,interdigitate)sothateachmaintainsitsfunction.

Positivecontrolrequiresaninteractionbetweenthebound

regulatoryproteinandpolymerase.Theregulatoryproteinmusthaveappropriateaminoacidsonitssurfaceinapositiontobindto polymerase. The surface that interacts with polymerase isdifferent from the surface that binds DNA. The functionsDNA-binding and positive controlcan be distinguished: twoproteinsmaybindidenticallywhileonlyonemanifestspositivecontrol.

FURTHERREADING:RELATEDREVIEWS

1.Hawley,D.K.andMcClure,W.R.(1983).CompilationandanalysisofEscherichiacolipromoterDNAsequences.Nucl.AcidsRes.11,2237-2255.

2.Maniatis,T.andPtashne,M.(1976).ADNAoperator-repressorsystem.Sci.Amer.234,64-76.

3.Pabo,C.O.andSauer,R.T.(1984).Protein-DNArecognition.Ann.Rev.Biochem.53,293-321.

4.Ptashne,M.,Johnson,A.D.,andPabo,C.O.(1982).Ageneticswitchinabacterialvirus.Sci.Amer.247,128-140.

5.Siebenlist,U.,Simpson,R.B.,andGilbert,W.(1980).EscherichiacoliRNApolymeraseinteractshomologouslywithtwodifferentpromoters.Cell20,269-281.

CHAPTERTHREE

CONTROLCIRCUITSSETTINGTHESWITCH

Manyvirusesgrowinonlyoneway.Soonafterinfectingthehost cell the viral genes work vigorouslynew proteins aresynthesized that extensively replicate the viral chromosomes,packagetheminnewviralparticles,andlysethecell.

Aswehavenoted,multipliesinsuchalyticfashion,butit also has an alternative. In a lysogenic bacterium the phagegenes required for lytic growth are turned off and the phagechromosome is replicated passively by proteins encoded andsynthesized by the bacterium. In the parlance ofChapterOnewesaythat themastercontrolelementtheswitchhasbeenset so that a single phage protein, the repressor, dominates.Recall that the repressor turns off the other phage genes as itturns on its own gene,cl. This otherwise stable situation isperturbedbyinducingagents,suchasultravioletlight,thatfliptheswitchbydestroyingrepressor,andlyticgrowthbegins.

This chapter is concerned primarily with two questionsconcerninggeneregulationin.Howdoesdecidewhethertogrowlyticallyorto

lysogenizeanewlyinfectedbacterium?Presumablyitisausefultricktomultiplysurreptitiously,aspartofalysogen,iftheconditionsforvigorouslyticgrowtharenotoptimal.

Howdoesregulateitsgenesasgrowthproceedsdowneither

ofitstwoseparatepathways?When the phage grows lytically following infection it

must,inanorderlyfashion,replicateandpackageitsDNAandthen lyse the cell, all the while preventing synthesis ofrepressor.Whenalysogenisinduced,afurthertaskisfacedbytheprophage about to begin lytic growth: an enzymemust besynthesizedthatreleasesexcisestheprophagefromthehostchromosome.

In contrast, when the phage begins the lysogenizationprocess it must synthesize the enzymes that integrate itschromosome into that of the host and begin synthesis ofrepressor, while preventing expression of the various lyticgenes.Inotherwords,theswitchofChapterOnemustbesetinthelysogenicposition.

The lysis-lysogeny decision is an instructive example ofhowtheenvironmentcaninfluencethechoiceofdevelopmentalpathways.Asweshallsee,thefirstfewstepsofgeneregulationthat occur upon infection are identicalwhether thephage isultimatelytolysethecellortolysogenizeit.Atthecriticalstepthestateofthehostissensedbyaphageregulatoryprotein,andsubsequent events are appropriately funneleddownoneof thetwopathways.

We shall see that whether is growing lytically or isestablishing lysogeny, its pattern of gene regulation isorganized as acascade: one regulatory protein typically turnson (or off) a block of genes; that block of genes typicallyincludes another regulatory gene whose product in turnactivates(orrepresses)asecondblockofgenes,andsoon.

We begin with a brief overview of the activities of aninfecting chromosome as it travels down the lytic or the

lysogenic path. We then examine the patterns of geneexpressioninmoredetail,andreturntothequestionofhowthelysisorlysogenydecisionismade.

ABRIEFOVERVIEWOFGROWTH

TheGeneticMap

AsimplifiedmapofthechromosomeisshowninFigure3.1.Only six genes are named individually in this representation:the regulatory genescl,cll,clll,N,cro, andQ.Theremaininggenesareindicatedingroupsaccordingtothefunctionsoftheproteinstheyencode.

Figure3.1. Thechromosome.Ingeneral,genesofrelatedfunctionaregroupedtogether. The genes within each of these groups are, as a rule, regulatedcoordinately. On this map six control genes are named individually, as are twosites,att(attachmentsite)andcos(cohesiveends).

The region named replication includes the two genesrequired for DNA replication. The region labelled lysisincludes three genes whose products lyse the bacterium. Therecombinationregioncontainssometengenes,includingtwowhoseproducts integrate thephage chromosome into thehostchromosome during lysogenization and excise it duringinduction.Theapproximatelytenheadgenesencodeproteinsthatconstructthephageheadandsome12morethephagetail.Wewillidentifysomeoftheseindividualgenesaswediscussgrowth.

Circularization

The genetic map is shown as a circle because the chromosome, a rod in the phage particle, circularizesimmediatelyuponbeing injected into thebacterium,asshowninFigure3.2.Theendsofthechromosomecalledcohesiveendsare joined by a bacterial enzyme, producing a pair ofcontinuous intertwined circularDNA strands. The joiningbringstogetherthelysisandthetailgenes.Attheendoflyticgrowth, when the new phage chromosomes are packaged intonewphageheads,theendsofthechromosomesareseparated.

Figure 3.2. Circularization of the chromosome. The sticky ends of the chromosome are 12 bases of single-stranded DNA that emerge, one from eachstrand, at the ends of the molecule. They pair spontaneously, and bacterialenzymeslinkthestrandstogethertoproduceacontinuouscirculardouble-strandedDNAmolecule.

Figur