分子生物學學習資料：MB answers 08-14

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Chapter8MajorShiftsinBacterialTranscription

SeeFigure8.2.WhenphageSPO1infectsBacillussubtilis,thefirststepinitstranscriptionprograminvolvestranscriptionofphageearlygenes,includinggene28bythehostpolymerase.Theproductofthisgene,gp28,isasigmafactorthatbindswiththehostcorepolymeraseresultinginaswitchfromthetranscriptionofhostgenesandphageearlygenestotranscriptionofphagemiddlegenes,includinggp33andgp34.Thesetwophagepolypeptides,gp33andgp34,constituteanothersigmafactor,whichassociateswiththehostpolymerasetodirectexpressionofthephagelategenes.

GeneticandbiochemicalstudiessupportthemodelofSPO1phagetranscriptionpresentedabove.Phagemutantsingene28losetheabilitytoswitchfromtranscriptionofearlygenestomiddlegenes.Mutationsineithergene33and34resultinlossofabilitytoswitchtranscriptionfrommiddletolategenes.Thissupportstheideathatgene28isneededfortheearlytomiddleswitchandgenes33and34areneededforthemiddletolateswitch.FurthersupportforthemodelhascomefromthepurificationbyPeroetal.ofanumberofRNApolymeraseactivitiesfromSPO1-infectedcells.Itwasdemonstratedthatoneofthese,whichcontainsgp28,isspecificforphagemiddlegenesandanother,whichcontainsgp33andgp34,isspecificforthephagelategenes.

InordertodemonstratethatB.subtilisErecognizesthesporulation-specific0.4kbpromoter,whereasArecognizesavegetativepromoter,wewouldsetupthefollowingexperiment.Wewoulddesignaplasmidcarryingavegetativepromoterandgene,bothcontainedwithinarestrictionfragmentofaparticularsize,say2kb.Theplasmidwouldalsocontainwithinaknownrestrictionfragmentofadifferentsize,say0.8kb,asecondgeneundercontrolofthe0.4kbsporulation-specificpromoter.Wewouldthenusetheseplasmidsinaninvitrotranscriptionassayusingradiolabelednucleotides,coreRNApolymeraseandeitherEorA.Inordertodeterminewhichgeneistranscribedwhenthepolymeraseisassociatedwiththedifferentsubunits,wewoulddoaSouthernblotandhybridizethelabeledtranscriptstorestrictiondigestsoftheplasmids.Theresultswewouldexpectareasfollows:ThetranscriptsgeneratedfromRNApolymeraseassociatedwiththeAsubunitwouldhybridizewiththe2kbrestrictionfragment.ThisisbecauseAspecifiestranscriptionofvegetative-specificgenes.ThetranscriptsgeneratedfromtheRNApolymeraseassociatedwiththeEsubunitshouldhybridizewiththe0.8kbrestrictionfragment.ThisisbecausethetranscriptionofthisDNAisdirectedbythe0.4kbsporulation-specificpromoterthatisrecognizedbyE.FortheactualexperimentalplanandresultsobtainedbyLosickandcolleagues,seeFigures8.4and8.5.

B.subtilisaltersitstranscriptionprogramduringsporulationbymeansofdifferentfactorswhichassociatewiththecoreRNApolymeraseanddirecttheexpressionofgenesthatarespecifictosporulation.Thepromotersequencesthatdirecttheexpressionofsporulation-specificgenesaredistinctfromthosepromotersequencesingenesrequiredforvegetativegrowth.Thesepromotersarerecognizedbydistinctfactors.Forexample,AspecificallyrecognizespromotersofgenesrequiredformaintenanceofthevegetativestateandAisnotproducedduringsporulation.Someofthesefactorsassociatedwithsporulationare,F,E,H,C,andK.ThefirstfactorproducedduringthesporulationprocessisFanditinitiatesaseriesofgeneticswitchesthatactinconcerttocontrolthesporulationtranscriptionalprogram.

SeeFigure8.6.RunofftranscriptioncanbeusedtodemonstratethatB.subtilusEspecificallytranscribesgenesundercontrolofthespoIIDpromoter.InsuchanexperimentwewoulduseaDNAfragmentcontainingaspoIIDpromoterdirectingaspoIIDgenethatistruncatedataknowndistancefromthetranscriptionstartsite.Thiswillallowustopredictthesizetranscriptthatwillbeproducedfromthisgene.Wewouldsetupaninvitrotranscriptionreactioninthepresenceofradiolabelednucleotidesandcorepolymeraseassociatedwithdifferentfactors.TheautoradiographwouldshowrunofftranscriptsonlywhenspoIIDistranscribedinthepresenceofE.

TheheatshockresponseinE.coliismediatedbyasigmafactor,32(H),aproductoftherpoHgene.Thepresenceof32allowsthetranscriptionofgenesencodingchaperoneswhichareinvolvedinrefoldingdenaturedproteins.Italsoleadstotranscriptionofgenesencodingproteaseswhichdegradeaberrantproteins.Whileincreasedtranscriptionof32isinitiateduponheatshock,alternativemechanismsexisttoallowamorerapidincreaseintheamountof32availableforactivationoftheheatshockgenes.Oneofthesemechanismsisincreasedstabilityof32afterheatshock.32isnormallyassociatedwithchaperoneswhichdestabilizetheprotein,butimmediatelyuponheatshockthesechaperonesbindalargenumberofcellularproteinsand32isleftfreetoactivateheatshockgenes.Furthermore,hightemperatureincreasestheefficiencywithwhichrpoHmRNAistranslated.Thisisasaresultofmeltingofsecondarystructureinthe5’regionofthemRNA.

ThephageT7hasarethreephaseoftranscriptioncalledI,IIandIII.Thereare5phageclassIgenesandthesearetranscribedbythehostRNApolymerase.OneoftheseencodesaphageRNApolymerase.ThisRNApolymeraseishighlyspecificandforclassIIandIIIgenes.

SeeFigure8.12.Bacteriophageaccomplishestheswitchfromimmediateearlytodelayedearlytolatetranscriptionbymeansofantiterminators.ThehostRNApolymerasetranscribestheimmediateearlygenes,croandN,usingthepromotersPRandPL.ThemRNAproducedbythesepromoterscanbeextendedtoincludethetranscriptsofthedelayedearlygenes;however,thisisblockedbyaterminatorsequencebetweentheimmediateearlygenesandthedelayedearlygenes.Nencodesanantiterminatorthatallowsthepolymerasetoreadthroughtheterminator.Croencodesaproteinthatsuppressesthelysogenicpathwaybyblockingthetranscriptionofthegene,cI,whichencodestherepressorandwhichisnecessaryformaintenanceoflysogeny.Thedelayedearlygenes,OandP,encodeproteinsnecessaryforphagereplication.Thethirddelayedearlygene,Q,isanotherantiterminatoranditsproductionallowstranscriptionofthelategenes.ThelategenesaretranscribedfromapromoterPR’.IntheabsenceofQ,transcriptionfromthispromoterishaltedafter194basesbecauseofaterminationsequence.ThepresenceofQallowsthepolymerasetobypassthisterminatorandthelategenesneededtoproducephageheadandtailproteinsaretranscribed.

SeeFigures8.13and8.14.KeytoN-directedantiterminationinphage-infectedE.colicellsisasitecallednut(N-utilization)immediatelydownstreamofPL.ThissiteisdistinctfromtheterminatorsequenceattheendoftheNgene.TheNproteinbindsthenutsiteonthemRNAtranscriptandacomplexofproteins,includingNusA,bindstothepolymerase.TheNproteinthenassociateswithNusAandthiscomplexofproteinsassociatedwiththenutsitealterthepolymerasesoitcanreadthroughtheterminatorattheendoftheNgene.

SeeFigure8.15.WhentheelongationcomplexreachestheendoftheNgeneitpausesduetothepresenceofastringofweakUAbasepairsintheRNA/templatehybrid.Duringthispausetheupstreamhalfofthehairpinloop-formingregionoftheRNAbindstotheupstreambindingsiteoftheRNApolymerase.Theupstreamhalfofthehairpin-formingregionoftheRNAcanbereleasedfromtheRNApolymeraseandahairpincanform.Hairpinformationcausestheelongationcomplextobedestabilizedandterminationoccurs.Thereforeterminationisheavilydependentonthespeedandefficiencyofhairpinformationwhiletheelongationcomplexispaused.ThisprocessisaffectedbyboththeNandtheNusAprotein.ThepresenceofNusAalonefacilitateshairpinformation.ItweakensthecontactsbetweentheupstreambindingsiteoftheRNApolymeraseandupstreamhalfofthepotentialhairpinintheRNA,allowingittobindtoitsotherhalf.Thisstimulatestermination.Nalsocontactstheupstreamhalfofthepotentialhairpin.BindingofNeliminatesthepositiveeffectsofNusAandhampersratherthanstimulateshairpinformation,thusinhibitingtermination.Intriguingly,NusAchangesitsrolewhenNispresent.InthepresenceofN,NusAcanadditionallybindtheupstreamhalfofthehairpinwhereitwillslowhairpinformationevenfurther.

SeeFigure8.16.AnexperimentthatdemonstratesthatbothNandNusAcontacttheRNAthatformsthefirstorupstreamhalfofthehairpinloopwouldsupportthehypothesisthatNandNusAcontrolhairpinformationattheintrinsicterminator.TodemonstratebindingofproteinstoRNA,UVcross-linkingexperimentscanbeused.WecanUVcross-linkalabeledRNAtoaproteinbyintroducingmodifiedbasesintotheRNA.WecanthenvisualizelabeledproteinsboundtotheRNAusingPAGEandautoradiography.Insuchanexperiment,wecanuseamutantterminatorthatproducesaslow-movingelongationcomplex.Thiswillfacilitatecross-linkingoftheRNAtotheproteinsNusAandN.SubstrateanalogscanbeincorporatedintotheRNAchainbywalkingtheelongationcomplexdownthetemplate.InthismannerwecanintroduceanalogsatpositionsintheRNAcorrespondingtotheupstreamorthedownstreamportionofthehairpinloop.ThisRNAbeincubatedwitheitherN,NusA,orboth,andcanbesubsequentlyUV-irradiatedtoallowcross-linkingofanyproteinboundtoregionsoftheRNAcontainingtheribonucleotideanalogs.WewouldexpectthedatatoshowthateitherNusA,N,orbothtogether,canbecross-linkedtonucleotidesinthe–24positioncorrespondingtotheupstreamhalfofthehairpinloop.However,theproteinscannotbeboundtoposition–14,correspondingtothedownstreamhalfofthehairpinloop.Thesedata,obtainedbyGusarovandNudler,supportthehypothesisthatbothNandNusAcontacttheRNAthatformsthefirstorupstreamhalfofthehairpinloop.AsimilarexperimentbyGusarovandNudlershowedthatNstimulatedbindingbetweentheupstreamhalfofthehairpinandsubunitsand’oftheRNApolymerase;furthermore,NandNusAstimulatedthisbindingevenmore,inaccordwiththehypothesisthatNandNusAcollaboratetopreventhairpinformation,andtherebypreventterminationoftranscriptionatintrinsicterminators.

SeeFigure8.18.DespiteitsinabilitytorecognizeRNApolymerase,thePREpromotercandirecttranscriptionoftheCIgene.ThisisbecauseCIIbindstotheweak-35boxofthepromoterandhelpsRNApolymerasebind,thusallowingtranscriptiontoproceed.

ThebasescontactedbyRNApolymeraseinregionthe-35boxofthePREpromoterareontheoppositesideoftheDNAdoublehelixfromthosebasesinthesameregionoftheDNAthatarecontactedbyCII.Thus,byapproachingtheDNAfromoppositesides,CIIandRNApolymerasecanbindtothesameregionofDNAsimultaneously.

SeeFigures8.20and8.21.Therepressorregulatesitsownsynthesisbothpositivelyandnegativelyasfollows.Inalysogen,repressordimersbindpreferentiallytoOR1andOR2.ThisbindingactivatesexpressionfromPRMthuspositivelyregulatingexpressionofrepressorfromcI.Astherepressorconcentrationincreases,OR3bindingsitesarefilledandthisinactivatesexpressionfromPRMthusnegativelyregulatingcIexpression.Aninvitrorun-offtranscriptionassaycanbeusedtodemonstratethisphenomenon.ADNAtemplateisengineeredsuchthatitcontainstheORregioncontrollingleftwardexpressionofcIfromPRMandrightwardexpressionofcrofromPR.Wecansynthesizelabeledtranscriptsinthepresenceofincreasingamountsofrepressor.InsuchanexperimentweexpecttoobservethatatlowconcentrationsofrepressorthereisastimulationofrepressorproductionfromPRMbutatahigherconcentrationofrepressorthereisadecreaseinrepressorproductionfromPRM.Also,ifwemonitorthetranscriptionofcrointhisexperimentwewillseeinhibitionofcrotranscriptionatlowrepressorconcentrationsandanabolitionofcrotranscriptionathigherrepressorconcentration.

SeeFigure8.23.

SeeFigure8.24.AnintergenicsuppressorexperimentcanbeusedtoshowthattherepressorinteractswiththesubunitoftheE.coliRNApolymerase.Insuchanexperiment,wewouldusearepressormutantthatcannotstimulatetranscriptionfromthePRMpromoter.Ifthislossofactivityisduetoalossoftherepressor’sabilitytointeractwithRNApolymeraseatthepromoter,weshouldbeabletoisolateanE.colistrainwithacompensatingmutationintheRNApolymeraseallowingtheinteractiontooccurandrestoringtranscriptionfromthePRMpromoter.Suchmutationsarerare;however,wecanuseapositiveselectionscreentoidentifyonlythedesiredmutations.Wewouldsetuptheexperimentasfollows.Usingaprophage,wecanintroduceintoE.coliacIgenebearingamutationthatpreventsrepressor’sinteractionwithRNApolymeraseatthePRMpromoter.ToscreenforcompensatingmutantsinRNApolymerase,wecanintroduceasecondprophagecarryingakanamycinresistancegene(kanR)underthedirectionofthePRMpromoter.BygrowingtheseE.colionkanamycin,weallowonlysurvivalofcellswithasuppressormutationinRNApolymerase.ThemutationallowstranscriptionofthekanRgeneduetorestorationoftheinteractionbetweentheRNApolymeraseandtherepressor.TodemonstratethattheinteractionbetweenRNApolymeraseandtherepressorinvolvesthe-subunitofRNApolymerasewecanintroduceintothecellswiththekanRgeneconstruct,acollectionofmutantrpoDgenesencodingthe-subunit.KanamycinresistantcelllinescarryingamutantrpoDgeneprovideevidencethattheinteractionbetweenRNApolymeraseandrepressorinvolvesthe-subunit.

SeeFigure8.26.ThedecisiontogrowlysogenicallyorlyticallyisdeterminedbytherelativeamountsoftheproductsofthegenescroandcIwithinthecell.cIproducestherepressor,thepresenceofwhichdetermineslysogenyandisincompatiblewiththelyticcycle.ThecroproteininhibitstranscriptionfromthecIgenewhileatthesametimeinducingtranscriptionofitsowngeneandthegenesnecessaryforthelyticcycle.IfcIpredominates,lysogenyensues;ifcropredominates,lysogenyisinhibitedandthelyticcycleensues.TwopromotersimportantincontrollingtheexpressionofthesegenesarePRMandPR.ThepromoterPRMdirectsleftwardtranscriptionofcI,andthepromoterPRdirectsrightwardtranscriptionofcro.ThestrugglebetweencIandcroforlysogenicorlyticinfectionispartlyareflectionofthefactthatthesametripartiteoperatorcontrolstheexpressionfrombothofthesepromoters.BothcroandcItargettheoperatorwhichhasthreebindingsites,OR1,OR2andOR3.ThecIproteinhasitshighestaffinityforOR1andrepressorboundtoOR1leadstoco-operativebindingatOR2.IfrepressorispresentitbindstoOR1andOR2andexpressionofcIfromthePRMpromoterisinduced.Thustherepressormaintainsitsownexpression.WhileexpressionfromPRMisinduced,thereisaconcomitantinhibitionofexpressionofcrofromPR.IfontheotherhandthecroproteinpredominatesitbindspreferentiallyatOR3andthispreventsbindingofRNApolymerasetoPRMthuspreventingrepressorsynthesis.TranscriptionfromPRisstimulatedandthegenesforthelyticcycleareinduced.ItisapparentthereforethatthekeytothestruggleistherelativelevelsofthecroandcIgeneproducts.Thisisdeterminedbyenvironmentalfactors.Anabundanceofnutrientsfavorsthelyticcycleandalessfavorablenutritionalenvironmentfavorslysogeny.Thisallowsthephagetoessentiallyputaholdonitslifecycleuntilmorefavorableconditionsprevail.TheproductofthegenecIImediatesthesensingofnutrientconditions.CIIissusceptibletodegradationbyproteasesandtheseenzymesareabundantinnutrient-richmedia.LowlevelsofCIIarethereforepresentinnutrient-richmediaandhigherlevelsarepresentinnutrient-poormedia.CIIfavorslysogenybyactivatingthePREpromoterwhichproducestherepressor.CIIadditionallyfavorslysogenybecausePREpromotergeneratesanti-sensecrotranscripts.

SeeFigure8.27.MutagenicinsultssuchasUVlightorchemicalmutagensreleaseλphagefromlysogenyasaresultofinductionoftheSOSpathway.AkeyplayerinthispathwayisRecA.RecAnormallyplaysaroleinrecombinationbutitalsoactsasaco-proteaseinducingalatentproteaseactivityintheλrepressor.Thisleadstocleavageandinactivationoftherepressor.Astherepressorisdestroyeditcannolongerinduceitsownexpressionandtheoperatorsitestowhichitbinds,OR1andOR2,arevacated.ThisallowsthepolymerasetobindtoPRandcroistranscribed.TheproductofthecrogenethenbindstoOR3andstimulatesisownexpressionwhileinhibitingexpressionofλrepressor.Thegenesdownstreamofcrothatareneededforthelyticcyclearealsotranscribed.Inthismannerthephageshiftsfromalysogenicstatetoalyticstate.

AnalyticalQuestions

Inordertestthehypothesisthataparticulargenehastwodifferentpromotersthatarerecognizedbytwodistinct-factors,thefollowingapproachcanbeused.Wecanpurifypolymeraseactivitiesfromcellsinwhichthisgeneisactive.Ifweidentifytwopolymeraseactivitiesby,forexample,DNA-cellulosechromatographywecanusethesepolymerasesinarun-offtranscriptionassaywithradiolabelednucleotides.Wewoulduseapolymeraseassociatedwithone-factorinoneassayandapolymeraseassociatedwithasecond-factorinasecondassay.Asatemplatewecanuseatruncatedclonedgenefragmentandifweobserverun-offtranscriptsoftwodifferentsizesproducedbythetwodifferent-factors,thissupportsthehypothesisthatthegenecontainstwopromoters.

AnE.colicellthatislysogenicforaparticularphageisimmunetosuperinfectionbyanidenticalstrainof.Thisisbecausetheprophageisproducingtherepressor(CI)andthisrepressorwillturnoffallphagegenesexceptthegeneencodingtherepressoritself.TherepressormoleculespresentintheE.coliuponinfectionbythesecondphage,willinactivateallthegenesintheincomingphagethusrenderingitincapableofsuperinfectingthelysogen.

WerewetotransfectE.colicellslysogenizedwithaparticularphagewithasecondphagehavingoperatorsequencessignificantlydifferentfromthatofthelysogen,wewouldexpecttogetsuperinfection.Thisisbecausetherepressorproducedbythelysogenwillnotrecognizetheoperatorsfromthesecondphage,resultinginnorepressionandallowinggeneexpressioninthesecondphage,establishinganinfection.

WerewetoirradiategeneticallydifferentE.coliλlysogensandonebecamealyticstrainandoneremainedlysogenicwecouldexplainthisphenomenonbyhypothesizingthatthenon-lyticstrainhadamutationintheRecAgene.AmutationsuchasthiswouldpreventrecA-induceddestructionoftheλrepressor,meaningthatE.coliwouldnotbereleasedfromlysogeny.

InastrainofinphagethatismutantinthecIIgenelyticinfectionswouldoccur100%ofthetime.CIIisneededtoactivateexpressionfromPRE,thepromoterforrepressorestablishment.Initsabsencenoλrepressorissynthesizedtoinducelysogeny.Additionally,CIIinducesexpressionoftheQandcrogenestheproductsofwhichareneededfortranscriptionofthelategenesrequiredforthelyticcycle.Alternatively,thephagecouldcarryamutationinthecIgene,whichencodestherepressor.Withouttherepressor,therecanbenolysogenicinfection.

Astrainofphagethatalwaysproduceslysogenicinfectionsislikelytobemutatedinthegenecro.ThecroproteininhibitstranscriptionfromthecIgeneencodingtheλrepressorwhileatthesametimeinducingtranscriptionofitsowngeneandthegenesnecessaryforthelyticcycle.

AstrainofphagewithaninactivatedNgenewouldproduceneitherlyticinfectionsnorlysogenicinfections.ThisisbecauseNfunctionsasanantiterminatortoallowexpressionofthedelayedearlygenes.Theproductsofthedelayedearlygenesarerequiredforestablishmentofboththelyticcycleandthelysogeniccycle.

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Chapter9DNA-ProteinInteractionsinProkaryotes

SeeFigure9.2:

SeeFigures9.3and9.4.Aminoacidswappingexperimentscanbeusedtoshowwhichaminoacidsareimportantinspecifyingbindingofrepressorstotheiroperators.Intheseexperiments,aminoacidresiduesthatarepredictedbystructuralstudiestoplayaroleinbindingaparticularoperatorarealteredbysite-directedmutagenesis.Tousetheexamplegiveninthetextoftheλ-likephages434andP22,wewouldpredictthatifweweretoswaptheaminoacidswehypothesizeasbeingimportantforoperator-bindingintheP22repressorforthosehypothesizedtoperformthesamefunctioninphage434,theengineeredP22phagerepressorwouldnowbind434operators.Inotherwords,thekeyaminoacidsinoperatorbindingfromonerepressorcanconferthesameoperatorbindingspecificityonarepressorfromadifferentphage.WecanvisualizethechangeinDNAbindingspecificitiesbyDNasefootprintingexperiments.Wewouldexpecttoobservebindingofthealtered434repressortoP22operatorsandviceversa.Functionalstudiescanalsobeusedtodeterminethebindingspecificityrepressorstotheiroperators.E.colilysogenicforphage434(actually,lysogenicforalambdaphagebearingthe434immunityregion),forexample,willbeimmunetosuperinfectionbyanotherlambdaphagebearingthe434immunityregionbecausetherepressorinthelysogenwillinactivatethegenesintheincomingphage.Ifhowever,weweretoengineeralambdaphagewitha434immunityregionsuchthatitnowhadtheaminoacidsinitsrepressorbindingdomainthatarekeytobindingtheP22operators,alysogenforthisrecombinantphagewouldnolongerbeimmunetosuperinfectionbythelambdaphagebearingthe434immunityregion,butwouldbeimmunetosuperinfectionbylambdaphagebearingtheP22immunityregion.

TheλrepressorandCrobindwithdifferentaffinitiestodifferentregionsofthesameoperators.Specifically,repressorbindswiththehighestaffinitytoOR1andthelowestaffinitytoOR3whileCrobindswithitshighestaffinitytoOR3anditslowestaffinitytoOR1.Thesespecificitiesaremediatedbyspecificaminoacid-basepairinteractionsbetweentherecognitionhelixandthemajorgroveoftheDNA.ThecriticalaminoacidsandbasepairsaredifferentforCroandfortheλrepressor.

GlutamineandasparaginesidechainstendtomakehydrogenbondswithDNA.

MethyleneandmethylgroupsonaminoacidstendtobindtoDNAbyhydrophobicinteractions.

AhydrogenbondnetworkinthecontextofDNA-proteininteractionsreferstothephenomenonwherebyproteinscaninteractwithaDNAtarget,notonlybyhydrogenbondswiththeDNAbasepairs,butadditionallybyaminoacid-aminoacidbondsandaminoacid-phosphatebackbonebonds.Inmanycases,theattractionofoneaminoacidforanothercanindirectlyfacilitateDNAbindingbypositioninganaminoacidsidechainoptimallyforinteractionwithitstargetbasepair.Ahydrogenbondnetworkthereforeinvolvesatleastathree-wayinteractionbetweenaminoacidsandeithertheDNAbackboneorabasepair.SeeFigure9.8forexamples.

SeeFigure9.12b:

SeeFigure9.13

TherecognitionhelixofthelrepressorfitssidewaysintothemajorgrooveofitsoperatorDNAwhiletherecognitionhelixofthetrprepressorinsertsalmostatrightanglestothemajorgrooveofitsoperatorDNA.

TherecognitionsitesforbindingproteinsareoftenpresentintwoormorecopiesontheDNAmolecule.Thisallowsbindingofthetargetproteinsasdimersoroligomers.BindingaproteindimertoaduplicatedrecognitionsiteonaDNAmoleculerequireslessenergythatbindingoftwomonomersseparatelytotheDNA.BindingadimertoDNAismoresuccessfulthanbindingtwomonomerstoDNAbecausethesecondproteininthedimerisautomaticallyinthecorrectorientationforbindingtothesecondsite.WethereforegaintheenergyrequiredtoorientthesecondproteincorrectlywithrespecttoitsDNAtarget.

SeeFigure9.16:

+=increasedDNasesensitivity

-=decreasedDNasesensitivity

SeeFigure9.17.CooperativebindingofλrepressordimerstoDNAoperatorsseparatedbyanintegralnumberofhelicalturnscanbedemonstratedusingDNasefootprintingexperiments.Theprinciplebehindsuchexperimentsisasfollows.CooperativebindingofrepressordimerstotwodistantoperatorsonaDNAmoleculeisfacilitatedbyloopingoutoftheinterveningDNA.ThisloopingresultsinalternatingareasofincreasedanddecreasedsensitivitytoDNaseintheinterveningregion.Thisisaresultofcompressionofthebackboneontheinsideoftheloopandexpansionofthebackboneontheoutsideoftheloop.OnanautoradiographfromaDNasefootprintingexperiment,thiscanbevisualizedasastutteredbandingpatternintheregioncorrespondingtotheloop.ThisisinadditiontothetwoprotectedareascorrespondingtotherepressorbindingsitesIncontrast,non-cooperativebindingwillresultintwoprotectedareascorrespondingtotherepressorbindingsitesasbeforebuttherewillbenoalterationinthepatternofDNaseIsensitivityintheinterveningregion.Todemonstratethatcooperativebindingrequiresanintegralnumberofturnsseparatingthetworepressor-bindingsiteswewouldengineerDNAmoleculescontainingrepressor-bindingsitesseparatedbyvariousdistances.Onlydistancesthataremultiplesof10.5basepairswillcontainanintegralnumberofhelicalturns.TheseDNAmoleculesarelabeled,allowedtobindrepressordimersandthensubjectedtotreatmentwithDNase.Wewouldexpecttoobserveastutteredbandingpatternintheregionbetweentherepressorbindingsitesonlywhenthosesitesareseparatedbyanintegralnumberofturns.Sucharesultwouldsuggestthattheindividualrepressordimersarebindingcooperatively,facilitatedbyloopi