转录的初始化.ppt

Chapter20 Initiationoftranscription 20 1Introduction20 2EukaryoticRNApolymerasesconsistofmanysubunits20 3Promoterelementsaredefinedbymutationsandfootprinting20 4RNApolymeraseIhasabipartitepromoter20 5RNApolymeraseIIIusesbothdownstreamandupstreampromoters20 6ThestartpointforRNApolymeraseII20 7TBPisauniversalfactor20 8TBPbindsDNAinanunusualway20 9Thebasalapparatusassemblesatthepromoter20 10Initiationisfollowedbypromoterclearance20 11Aconnectionbetweentranscriptionandrepair20 12PromotersforRNApolymeraseIIhaveshortsequenceelements20 13Somepromoter bindingproteinsarerepressors20 14Enhancerscontainbidirectionalelementsthatassistinitiation20 15IndependentdomainsbindDNAandactivatetranscription20 16Thetwohybridassaydetectsprotein proteininteractions20 17Interactionofupstreamfactorswiththebasalapparatus Enhancerelementisacis actingsequencethatincreasestheutilizationof some eukaryoticpromoters andcanfunctionineitherorientationandinanylocation upstreamordownstream relativetothepromoter 20 1Introduction Figure20 1AtypicalgenetranscribedbyRNApolymeraseIIhasapromoterthatextendsupstreamfromthesitewheretranscriptionisinitiated Thepromotercontainsseveralshort 200bp Anenhancercontainingamorecloselypackedarrayofelementsthatalsobindtranscriptionfactorsmaybelocatedseveralkbdistant DNAmaybecoiledorotherwiserearrangedsothattranscriptionfactorsatthepromoterandattheenhancerinteracttoformalargeproteincomplex 20 1Introduction Amanitin morefullya amanitin isabicyclicoctapeptidederivedfromthepoisonousmushroomAmanitaphalloides itinhibitstranscriptionbycertaineukaryoticRNApolymerases especiallyRNApolymeraseII 20 2EukaryoticRNApolymerasesconsistofmanysubunits Figure20 2EukaryoticRNApolymeraseIIhas 10subunits 20 2EukaryoticRNApolymerasesconsistofmanysubunits Cotransfectionisthesimultaneoustransfectionoftwomarkers 20 3Promoterelementsaredefinedbymutationsandfootprinting Figure20 3Promoterboundariescanbedeterminedbymakingdeletionsthatprogressivelyremovemorematerialfromoneside WhenonedeletionfailstopreventRNAsynthesisbutthenextstopstranscription theboundaryofthepromotermustliebetweenthem 20 3Promoterelementsaredefinedbymutationsandfootprinting Figure20 4TranscriptionunitsforRNApolymeraseIhaveacorepromoterseparatedby 70bpfromtheupstreamcontrolelement UBF1bindstobothregions afterwhichSL1canbind RNApolymeraseIthenbindstothecorepromoter Thenatureoftheinteractionbetweenthefactorsboundattheupstreamcontrolelementandthoseatthecorepromoterisnotknown 20 4RNApolymeraseIhasabipartitepromoter PreinitiationcomplexineukaryotictranscriptiondescribestheassemblyoftranscriptionfactorsatthepromoterbeforeRNApolymerasebinds 20 5RNApolymeraseIIIusesbothdownstreamandupstreampromoters Figure20 5Deletionanalysisshowsthatthepromoterfor5SRNAgenesisinternal initiationoccursafixeddistance 55bp upstreamofthepromoter 20 5RNApolymeraseIIIusesbothdownstreamandupstreampromoters Figure20 6PromotersforRNApolymeraseIIImayconsistofbipartitesequencesdownstreamofthestartpoint withboxAseparatedfromeitherboxCorboxB Ortheymayconsistofseparatedsequencesupstreamofthestartpoint Oct PSE TATA 20 5RNApolymeraseIIIusesbothdownstreamandupstreampromoters Figure20 7InitiationviatheinternalpolIIIpromotersinvolvestheassemblyfactorsTFIIIAandTFIIIC theinitiationfactorTFIIIB andRNApolymeraseIII 20 5RNApolymeraseIIIusesbothdownstreamandupstreampromoters TATAboxisaconservedA T richseptamerfoundabout25bpbeforethestartpointofeacheukaryoticRNApolymeraseIItranscriptionunit maybeinvolvedinpositioningtheenzymeforcorrectinitiation 20 6ThestartpointforRNApolymeraseII Figure20 8RNApolymerasesarepositionedatallpromotersbyafactorthatcontainsTBP 20 7TBPisauniversalfactor Figure20 9Aviewincross sectionshowsthatTBPsurroundsDNAfromthesideofthenarrowgroove TBPconsistsoftworelated 40 identical conserveddomains whichareshowninlightanddarkblue TheN terminalregionvariesextensivelyandisshowningreen ThetwostrandsoftheDNAdoublehelixareinlightanddarkgrey PhotographkindlyprovidedbyStephenBurley 20 7TBPisauniversalfactor Figure20 10ThecocrystalstructureofTBPwithDNAfrom 40tothestartpointshowsabendattheTATAboxthatwidensthenarrowgroovewhereTBPbinds PhotographprovidedbyStephenBurley 20 7TBPisauniversalfactor Figure20 11AninitiationcomplexassemblesatpromotersforRNApolymeraseIIbyanorderedsequenceofassociationwithtranscriptionfactors 20 8Thebasalapparatusassemblesatthepromoter Figure20 12TwoviewsoftheternarycomplexofTFIIB TBP DNAshowthatTFIIBbindsalongthebentfaceofDNA ThetwostrandsofDNAaregreenandyellow TBPisblue andTFIIBisredandpurple PhotographkindlyprovidedbyStephenBurley 20 8Thebasalapparatusassemblesatthepromoter Figure20 13PhosphorylationoftheCTDbythekinaseactivityofTFIIHmaybeneededtoreleaseRNApolymerasetostarttranscription 20 8Thebasalapparatusassemblesatthepromoter Figure20 14MfdrecognizesastalledRNApolymeraseanddirectsDNArepairtothedamagedtemplatestrand 20 9Aconnectionbetweentranscriptionandrepair Figure14 28TheUvrsystemoperatesinstagesinwhichUvrABrecognizesdamage UvrBCnickstheDNA andUvrDunwindsthemarkedregion 20 9Aconnectionbetweentranscriptionandrepair Figure20 15TheTFIIHcoremayassociatewithakinaseatinitiationandassociatewitharepaircomplexwhendamagedDNAisencountered 20 9Aconnectionbetweentranscriptionandrepair Figure14 37AhelicaseunwindsDNAatadamagedsite endonucleasescutoneithersideofthelesion andnewDNAissynthesizedtoreplacetheexcisedstretch 20 9Aconnectionbetweentranscriptionandrepair CAATboxispartofaconservedsequencelocatedupstreamofthestartpointsofeukaryotictranscriptionunits itisrecognizedbyalargegroupoftranscriptionfactors 20 10PromotersforRNApolymeraseIIhaveshortsequenceelements Figure20 16Saturationmutagenesisoftheupstreamregionoftheb globinpromoteridentifiesthreeshortregions centeredat 30 75 and 90 thatareneededtoinitiatetranscription ThesecorrespondtotheTATA CAAT 20 10PromotersforRNApolymeraseIIhaveshortsequenceelements Figure20 17PromoterscontaindifferentcombinationsofTATAboxes CAATboxes GCboxes andotherelements 20 10PromotersforRNApolymeraseIIhaveshortsequenceelements Table20 17UpstreamtranscriptionfactorsbindtosequenceelementsthatarecommontomammalianRNApolymeraseIIpromoters 20 10PromotersforRNApolymeraseIIhaveshortsequenceelements Table20 17UpstreamtranscriptionfactorsbindtosequenceelementsthatarecommontomammalianRNApolymeraseIIpromoters 20 10PromotersforRNApolymeraseIIhaveshortsequenceelements Figure20 18AtranscriptioncomplexinvolvesrecognitionofseveralelementsintheseaurchinH2Bpromoterintestis BindingoftheCAATdisplacementfactorinembryopreventstheCAAT bindingfactorfrombinding soanactivecomplexcannotform 20 10PromotersforRNApolymeraseIIhaveshortsequenceelements Enhancerelementisacis actingsequencethatincreasestheutilizationof some eukaryoticpromoters andcanfunctionineitherorientationandinanylocation upstreamordownstream relativetothepromoter 20 11Enhancerscontainbidirectionalelementsthatassistinitiation Figure19 39Indirectend labelingidentifiesthedistanceofaDNAasehypersensitivesitefromarestrictioncleavagesite TheexistenceofaparticularcuttingsiteforDNAaseIgeneratesadiscretefragment whosesizeindicatesthedistanceoftheDNAaseIhypersensitivesitefromtherestrictionsite 20 11Enhancerscontainbidirectionalelementsthatassistinitiation Figure19 40TheSV40minichromosomehasanucleosomegap PhotographkindlyprovidedbyMosheYaniv 20 11Enhancerscontainbidirectionalelementsthatassistinitiation Figure20 19Anenhancercontainsseveralstructuralmotifs Thehistogramplotstheeffectofallmutationsthatreduceenhancerfunctionto 75 ofwildtype Bindingsitesforproteinsareindicatedbelowthehistogram 20 11Enhancerscontainbidirectionalelementsthatassistinitiation Figure20 16Saturationmutagenesisoftheupstreamregionoftheb globinpromoteridentifiesthreeshortregions centeredat 30 75 and 90 thatareneededtoinitiatetranscription ThesecorrespondtotheTATA CAAT 20 11Enhancerscontainbidirectionalelementsthatassistinitiation Figure20 20Anenhancermayfunctionbybringingproteinsintothevicinityofthepromoter AnenhancerdoesnotactonapromoterattheoppositeendofalonglinearDNA butbecomeseffectivewhentheDNAisjoinedintoacirclebyaproteinbridge AnenhancerandpromoteronseparatecircularDNAsdonotinteract butcaninteractwhenthetwomoleculesarecatenated 20 11Enhancerscontainbidirectionalelementsthatassistinitiation Figure20 21DNA bindingandactivatingfunctionsinatranscriptionfactormaycompriseindependentdomainsoftheprotein 20 12IndependentdomainsbindDNAandactivatetranscription Figure20 22TheGAL4proteinhasindependentregionsthatbindDNA activatetranscription 2regions dimerize andbindtheregulatorGAL80 20 12IndependentdomainsbindDNAandactivatetranscription Figure20 23TheabilityofGAL4toactivatetranscriptionisindependentofitsspecificityforbindingDNA WhentheGAL4DNA bindingdomainisreplacedbytheLexADNA bindingdomain thehybridproteincanactivatetranscriptionwhenaLexAoperatorisplacednearapromoter 20 12IndependentdomainsbindDNAandactivatetranscription Figure20 24TheactivatingdomainofthetatproteinofHIVcanstimulateinitiationifitistetheredinthevicinitybybindingtotheRNAproductofapreviousroundoftranscription Activationisindependentofthemeans 20 12IndependentdomainsbindDNAandactivatetranscription Figure20 25ThetwohybridtechniqueteststheabilityoftwoproteinstointeractbyincorporatingthemintohybridproteinswhereonehasaDNA bindingdomainandtheotherhasatranscription activatingdomain 20 12IndependentdomainsbindDNAandactivatetranscription Figure20 21DNA bindingandactivatingfunctionsinatranscriptionfactormaycompriseindependentdomainsoftheprotein 20 13Interactionofupstreamfactorswiththebasalapparatus Figure20 26Anupstreamtranscriptionfactormaybindacoactivatorthatcontactsthebasalapparatus 20 13Interactionofupstreamfactorswiththebasalapparatus Figure20 24TheactivatingdomainofthetatproteinofHIVcanstimulateinitiationifitistetheredinthevicinitybybindingtotheRNAproductofapreviousroundoftranscription Activationisindependentofthemeans 20 13Interactionofupstreamfactorswiththebasalapparatus Figure20 11Aninitiationcomplexassemblesatpromot