分子生物学6真核生物的转录及其调控课件

TranscriptioninEukaryotesTranscriptioninEukaryotes1ThethreeRNAPolymerasescharacterizationandfunctionMainFeaturesofeukaryotictranscription1.Themechanismofeukaryotictranscriptionissimilartothatinprokaryotes.2.Alotmoreproteinsareassociatedwiththeeukaryotictranscriptionmachinery,whichresultsinthemuchmorecomplicatedtranscription.ThethreeRNAPolymerasesMain23.

Threeeukaryoticpolymerasestranscribedifferentsetsofgenes.Theactivitiesofthesepolymerasesaredistinguishedbytheirsensitivitiestothefungaltoxinα-amanitin.4.Inaddition,eukaryoticcellscontainadditionalRNAPolsinmitochondriaandchloroplasts.3.Threeeukaryoticpolymerase3ThreeeukaryoticpolymerasesTypeLocationSubstrateα-amanitinRNAPolINucleoliMostrRNAsgeneInsensitiveRNAPolIINucleoplasmAllprotein-codinggenesandsomesnRNAgenesVerysensitiveRNAPolIIINucleoplasmtRNAs,5SrRNA,U6snRNAandothersmallRNAsModeratelysensitiveThreeeukaryoticpolymerasesTy4α-amanitinThesensitivitiesofthreekindsofRNApolymerasesonα-amanitinα-amanitinThesensitivitiesof5RNApolymerasesubunitsEacheukaryoticpolymerasecontains12ormoresubunits.–thetwolargestsubunitsaresimilartoeachotherandtotheβ’andβsubunitsofE.coliRNAPol.–ThereisoneothersubunitinallthreeRNAPolhomologoustoαsubunitofE.coliRNAPol.–Fiveadditionalsubunitsarecommontoallthreepolymerases.–EachRNAPolcontainsadditionalfourorsevenspecificsubunit.RNApolymerasesubunitsEacheu6RNApolymeraseactivities1.TranscriptionmechanismissimilartothatofE.colipolymerase.Direction:5’3’2.Differentfrombacterialpolymerasae,theyrequireaccessoryfactorsforDNAbindRNApolymeraseactivities1.Tr7TheCTDofRNApolII1.TheC-terminusofRNAPolIIcontainsastretchofsevenaminoacidsthatisrepeated52timesinmouseenzymeand26timesinyeast.2.Theheptapeptidesequencis:

Tyr-Ser-Pro-Thr-Ser-Pro-Ser3.Thisrepeatedsequenceisknownascarboxylterminaldomain(CTD)4.TheCTDsequencemaybephosphorylatedattheserinesandsometyrosinesTheCTDofRNApolII1.TheC-85.TheCTDisunphosphorylatedattranscriptioninitiation,andphosphorylationoccursduringtranscriptionelongationastheRNAPolIIleavesthepromoter(Invitroresults).6.Becauseittranscribesalleukaryoticprotein-codinggene,RNAPolIIisthemostimportantRNApolymeraseforthestudyofdifferentialgeneexpression.TheCTDisanimportanttargetfordifferentialactivationoftranscriptionelongation.5.TheCTDisunphosphorylated9分子生物学6真核生物的转录及其调控课件10RNAPolIgenes:theribosomalrepeatsRibosomalRNAGenes&nucleolus1.Acopyof18S,5.8Sand28SrRNAgenesisorganizedasasingletranscriptionunitineukayotes.A45SrRNAtranscript(~13000ntlong)isproducedduringtranscription,whichisthenprocessedinto18S,5.8Sand28SrRNA.2.Pre-rRNAtranscriptionunitsarearrangedinclustersinthegenomeaslongtandemarraysseparatedbynontranscribedspacersquences.RNAPolIgenes:RibosomalRNA113.ContinuoustranscriptionofmultiplecopiesofrRNAgenesbyRNAPolIisessentialtoproducesufficientrRNAswhicharepackagedintoribosomes.4.ThearraysofrRNAgenes(rRNAcluster)looptogethertoformthenucleolusandareknownasnucleolarorganizerregions.5.DuringactiverRNAsynthesis,thepre-rRNAtranscriptsarepackagedalongtherRNAgenes,visualizingintheelectronicmicroscopeas“Christmastreestructures”.3.Continuoustranscriptionof12分子生物学6真核生物的转录及其调控课件13分子生物学6真核生物的转录及其调控课件14分子生物学6真核生物的转录及其调控课件15RNAPolIpromoters1.Generallyconsistsofabipartitesequenceintheregionprecedingthestartsite,includingcoreelementandtheupstreamcontrolelements(UCE).2.RNAPolIpromotersinhumancellsarebestcharacterized.•Coreelement:-45to+20,sufficientfortranscriptioninitiatiation.•UCE:-180to-107,toincreasethetranscriptionefficiency.•BothregionsarerichinG:C,with~85%identity.RNAPolIpromoters1.Generall16Upstreambindingfactor(UBF)AspecificDNA-bindingproteinthatbindstoUCE,aswellasadifferentsiteintheupstreamofthecoreelement,causingtheDNAtoloopbetweenthetwosites.(twobindingsiteshavenoobvioussimilarity)•UBFisessentialforhighleveloftranscription,andlowlevelofexpressionoccursinitsabsence.Upstreambindingfactor(UBF)A171.Doesnotbindtopromotersbyitself2.BindstoandstabilizestheUBF-DNAcomplex.3.Interactswiththefreedownstreampartofthecoreelement.4.RecruitRNAPolItobindandtoinitiatethetranscription.Selectivityfactor1(SL1)1.Doesnotbindtopromoters18SL1consistsof4proteins1.

TBP(TATA-bindingprotein)

afactoralsorequiredforinitiationbyRNAPolIIandIII.AcriticalgeneralfactorineukaryotictranscriptionthatensuresRNAPoltobeproperlylocalizedatthestartpoint.2.OtherthreesubunitsarereferredtoasTBP-associatedfactors(TAFIs)thatarespecificforRNAPolItranscription.SL1consistsof4proteins19分子生物学6真核生物的转录及其调控课件20OtherrRNAgenesInasimpleeukaryote,Acanthamoeba,therRNAgeneshaveonlyonecontrolelement(promoter)around12-72bpupstreamfromthetranscriptionstartsite.SimpleinitiationTIF-1(homologofSL-1)bindstothepromoter

RNAPolIbind

TIF-1remainsboundandtheRNAPolIisreleasedforelongation.OtherrRNAgenesInasimpleeu21RNAPolIIIgenes:5SandtRNAtranscriptionRNAPolIII1.Containsatleast16ormoresubunits2.Islocatedinnucleoplasm3.Synthesizestheprecursorsof5SrRNA,thetRNAsandothersmallnuclearandcytosolicRNAsRNAPolIIIgenes:RNAPolIII122PromotersforRNApolymeraseIIIMayconsistofbipartitesequencesdownstream

ofthestartpoint,withboxAseparatedfromeitherboxCorboxB.Ortheymayconsistofseparatedsequencesupstream

ofthestartpoint(Oct,PSE,TATA).PromotersforRNApolymeraseI23tRNAgenes1.TheinitialtranscriptsoftRNAgenesneedtobeprocessedtoproducethematuretRNA.2.ThetranscriptioncontrolregionsoftRNAliesafterthestartsitewithinthetranscribedregion.ThetwohighlyconservedcontrolsequencesarecalledAbox(5’-TGGCNNAGTGG)Bbox(5’-GGTTCGANNCC)AboxandBboxalsoencodeimportantsequencesinthetRNAitself,theD-loopandTΨC-loop.•Therefore,thehighlyconservedsequenceintRNAsarealsohighlyconservedpromoterDNAsequences.tRNAgenes1.Theinitialtrans24分子生物学6真核生物的转录及其调控课件253.TwocomplexDNA-bindingfactorsrequiredfortRNAtranscriptioninitiationTFIIIC---bindstoboththeAandBboxes,anassemblyfactorforpositioningTFIIIB.TFIIIB

(1)binds50bpupstreamfromtheAbox,buthasnosequencespecificityandthebindingpositionisdeterminedbytheDNAboundTFIIIC.(2)consistsofthreesubunits,oneofwhichisTBP,thegeneralinitiationfactor;thesecondiscalledBRF(TFIIB-relatedfactor);andthethirdiscalledB”.3.TwocomplexDNA-bindingfac26分子生物学6真核生物的转录及其调控课件275SrRNAgenes1.Tandemlyarrangedinagenecluster.(Inhuman,thereisasingleclusterofaround2000genes.)2.Transcriptioncontrolregions(promoters)areorganizedsimilartothoseoftRNA,exceptthatCboxisinplaceofBbox.

Cbox:+81-99bp;Abox:+50-655SrRNAgenes1.Tandemlyarran283.TranscriptionfactorsTheCboxactsasthebindingsiteforTFIIIA.TFIIIAactsasanassemblyfactorwhichallowsTFIIIC

tointeractwiththe5SrRNApromoter.TheAboxmayalsostabilizeTFIIICbinding.TFIIICisthenboundtoDNAsitenear+1.TFIIIBandTFIIICinteracttorecruitRNAPolIIItoinitiatetranscription.3.Transcriptionfactors29分子生物学6真核生物的转录及其调控课件30AlternativeRNAPolIIIpromotersManyRNAPolIIIgenesalsorelyonupstreamsequences

forregulationoftheirtranscriptione.g.U6snRNAandEpstein-BarrvirusUseonlyregulatorygenesupstreamfromtheirtranscriptionstartsites.AlternativeRNAPolIIIpromot31U6snRNA1.ThecodingregioncontainsacharacteristicAboxthatisnotrequiredfortranscription.2.TheupstreamsequencecontainssequencestypicalofRNAPolIIpromoters,includingaTATAboxatbases–30to–23.3.SharesseveralothertranscriptionfactorbindingsequenceswithmanyURNAgeneswhicharetranscribedbyRNAPolIISuggestion:commontranscriptionfactorscanregulatebothRNAPolIIandPolIIIgenesU6snRNA32RNAPolIIIterminationTheRNApolymerasecanterminatetranscriptionwithoutaccessoryfactors.AclusterofAresidueisoftensufficientfortermination.Xenopusborealisterminator:

5’-GCAAAAGC-3’RNAPolIIIterminationTheRNA33RNAPolIIgenes:promotersandenhancersRNAPolII1.locatedinnucleoplasm2.catalyzingthesynthesisofthemRNAprecursorsforallprotein-codinggenes.3.RNAPolⅡ-transcribedpre-mRNAsareprocessedthroughcapaddition,poly(A)tailadditionandsplicing.RNAPolIIgenes:RNAPolII1.34Promoters•MostpromoterscontainasequencecalledtheTATAboxaround25-35bpupstreamfromthestartsiteoftranscription.Ithasa7bpconsensussequence

5’-TATA(A/T)A(A/T)-3’•TBPbindstoTATAboxthatincludesanadditionaldownstreambp(8bp).Promoters•Mostpromotersconta35-TATAboxactsinasimilarwaytoanE.colipromoter–10sequencetopositiontheRNAPolIIforcorrecttranscriptioninitiation.ThespacingbutnotthesequencebetweentheTATAboxandthestartsiteisimportant.Transcriptionstartswithanadenine~50%ofthetime.-TATAboxactsinasimilarw36-SomeeukaryoticgenescontainaninitiatorelementinsteadofaTATAbox.Theinitiatorelementislocatedaroundthetranscriptionstartsite.-OthergeneshaveneitheraTATAboxnoraninitiatorelement,andusuallyaretranscribedatverylowrates.-Someeukaryoticgenescontai37Upstreamregulatoryelements•Thebasalelements(theTATAboxandinitiatorelements):primarilydeterminethelocationofthestartpoint,andsponsorinitiationonlyataratherlowlevel.•Upstreamregulatoryelements(URE)suchastheSP1boxandCCAATboxes,greatlyincreasethefrequencyofinitiation.UREislocatedwithin100-200bpfromthepromoter,andplaysanimportantroleinensuringefficienttranscription.Upstreamregulatoryelements•T38EnhancersSequenceelementswhichcanactivatetranscriptionfromthousandsofbasepairsupstreamordownstream.GeneralcharacteristicsofEnhancers1.

Exertstrongactivationoftranscriptionofalinkedgenefromthecorrectstartsite.EnhancersSequenceelementswhi392.activatetranscriptionwhenplacedineitherorientationwithrespecttolinkedgenes3.Abletofunctionoverlongdistancesofmorethan1kbwhetherfromanupstreamordownstreampositionrelativetothestartsite.4.Exertpreferentialstimulationoftheclosestoftwotandempromoters2.activatetranscriptionwhen40分子生物学6真核生物的转录及其调控课件41GeneraltranscriptionfactorsandRNAPolⅡinitiationTFIIDMultiproteinComplex,includingTBP,otherproteinsareknownasTAFIIs.TBPistheonlyproteinbindstoTATAboxTBP1.ageneraltranscriptionfactorboundtoDNAattheTATAbox.2.ageneraltranscriptionrequiredbyall3RNApol.3.hasasaddlestructurewithanoveralldyadsymmetry.GeneraltranscriptionfactorsT42TFIIA•bindstoTFIID•stabilizesTFIID-DNAcomplex•containsatleast3subunitsTFIIB&RNAPolbinding•bindstoTFIID•BindstoRNAPolwithTFIIFTFIIE,TFIIJ,TFIIHbinding

NecessaryfortranscriptionTFIIATFIIB&RNAPolbindi43分子生物学6真核生物的转录及其调控课件44分子生物学6真核生物的转录及其调控课件45phosphorylationofthepolymeraseCTDbyTFIIHFormationofaprocessiveRNApolymerasecomplexandallowstheRNAPoltoleavethepromoterregion.phosphorylationofthepolymer46TheinitiatortranscriptioncomplexForTATA-boxlackingRNAPolIIpromoters,TBPisrecruitedtotheinitiatorelement0verlappingthestartsitebysomeDNA-bindingproteins,TBPthenrecruittheothertranscriptionfactorsandpolymerasesimilartoTATAboxgenetranscription.Theinitiatortranscriptionco47分子生物学6真核生物的转录及其调控课件48分子生物学6真核生物的转录及其调控课件49Transcriptionofasinglegenemayberegulatedbymanydifferentfactorsinteractingwithregulatoryelementsupstreamordownstreamofthetranscribedsequence.

GeneXStartsite+1Regulatoryelements

tobindtranscriptionfactorsEukaryoticTranscriptionFactorsTranscriptionofasinglegene50Commonfeaturesoftranscriptionfactors1.bindspecificallytosomeDNAsites:specificmotifsinpromoters,upstreamregulatoryelements(UREs)orenhancerregions.Somefactorsmodulatetranscriptionbyprotein-proteininteraction2.Activate/represstranscription.Commonfeaturesof51Theactivityofatranscriptionfactorcanbeassignedtoseparateproteindomainsactivationdomains.DNA-bindingdomains.dimerizationdomains.Manytranscriptionfactorsoccurashomo-orheterodimers,heldtogetherbydimerizationdomains.ligand-bindingdomains.Allowingregulationoftranscriptionfactoractivitybybindingofanaccessorysmallmolecule.Thesteroidhormonereceptorsareanexamplecontainingallfourofthesetypesofdomain.Theactivityofatranscriptio52Domainswapexperiments

movingdomainsamongproteins,provingthatdomainscanbedissectedintoseparatepartsoftheproteins.

TheexperimentoffusingactivationdomainsofyeasttranscriptionfactorsGal4andGcn4intothebacterialLexArepressorTranscriptionactivationdomainsareseparablefromtheirDNAbindingactivity.DomainswapexperimentsThee53分子生物学6真核生物的转录及其调控课件54分子生物学6真核生物的转录及其调控课件55DNA-bindingdomainsHelix-turn-helixdomainDNA-bindingdomainsHelix-turn-56Examplesofhelix-turn-helixdomains1.Homeodomain:encodedbyasequencecalledthehomeobox,containinga60-amino-acid.IntheAntennapediatranscriptionfactorofDrosophila,thisdomainconsistsoffourα-helicesinwhichhelicesⅡandⅢareatrightanglestoeachotherandareseparatedbyacharacteristicβ-turn.Examplesofhelix-turn-helixd572.OtherDNA-bindingproteinssuchasthephageλcrorepressor,lacandtrprepressors,andcAMPreceptorprotein,CRP.Therecognitionhelix

ofthedomainstructureliespartlyinthemajorgrooveandinteractswiththeDNA.

TherecognitionhelicesoftwohomeodomainfactorsBicoidandAntennapediacanbeexchanged,andthisswapstheirDNA-bindingspecificities.2.OtherDNA-bindingproteins58ThezincfingerdomainThezincfingerdomain59ZincfingerdomainexistsintwoformsC2H2zincfinger:aloopof12aminoacidsanchoredbytwocysteineandtwohistidineresiduesthattetrahedrallyco-ordinateazincion.Thismotiffoldsintoacompactstructurecomprisingtwoβ-strandsandoneα-helix.Theα-helixcontainingconservedbasicaminoacidsbindsinthemajorgrooveofDNAZincfingerdomainexistsint60分子生物学6真核生物的转录及其调控课件61分子生物学6真核生物的转录及其调控课件62

Examples

(1)TFIIIA,theRNAPolIIItranscriptionfactor,withC2H2zincfingerrepeated9times.(2)SP1,with3copiesofC2H2zincfinger.Usually,threeormoreC2H2zincfingersarerequiredforDNAbinding.

Examples63C4zincfinger:zincioniscoordinatedby4cysteineresidues.Example

steriodhormonereceptortranscriptionfactors,consistingofhomo-orhetero-dimers,inwhicheachmonomercontainstwoC4zincfinger.C4zincfinger:zincionisco64分子生物学6真核生物的转录及其调控课件65ThebasicdomainRichinbasicaminoacidresiduesfoundinanumberofDNA-bindingproteinsgenerallyassociatedwithoneorotheroftwodimerizationdomains,theleucinezipperorthehelix-loop-helix(HLH)motif,resultinginbasicleucinezipper(bZIP)orbasicHLHproteins.DimerizationoftheproteinsbringstogethertwobasicdomainswhichcantheninteractwithDNA.ThebasicdomainRichinbasic66DimerizationdomainsLeucinezipperLeucinezipperproteinscontainahydrophobicleucineresidueateveryseventhpositioninaregionthatisoftenattheC-terminalpartoftheDNA-bindingdomain.Theseleucinesareresponsiblefordimerizationthroughinteractionbetweenthehydrophobicfacesoftheα-helices.Thisinteractionformsacoiled-coilstructureDimerizationdomainsLeucinezi67bZIP(basicleucinezipper)transcriptionfactors:

containabasicDNA-bindingdomainN-terminaltotheleucinezipper.TheN-terminalbasicdomainsofeachhelixformasymmetricalstructureinwhicheachbasicdomainsliesalongtheDNAinoppositedirection,interactingwithasymmetricalDNArecognitionsitewiththezipperedproteinclampbZIP(basicleucinezipper)tr68分子生物学6真核生物的转录及其调控课件69分子生物学6真核生物的转录及其调控课件70TheleucinezipperisalsousedasadimerizationdomaininproteinscontainingDNA-bindingdomainsotherthanthebasicdomain,includingsomehomeodomainproteins.Theleucinezipperisalsouse71Thehelix-loop-helixdomain(HLH)Theoverallstructureissimilartotheleucinezipper,exceptthatanonhelicalloopofpolypeptidechainseparatestwoα-helicesineachmonomericprotein.HydrophobicresiduesononesideoftheC-terminalα-helixallowdimerization.ExampleMyoDfamilyofproteins.

Thehelix-loop-helixdomain(H72Similartoleucinezipper,theHLHmotifisoftenfoundadjacenttoabasicdomainthatrequiresdimerizationforDNAbinding.BasicHLHproteinsandbZIPproteinscanformheterodimersallowingmuchgreaterdiversityandcomplexityinthetranscriptionfactorrepertoire.Similartoleucinezipper,the73分子生物学6真核生物的转录及其调控课件74分子生物学6真核生物的转录及其调控课件75TranscriptionactivationdomainsAcidicactivationdomainsRichinacidicaminoacidsExistsinmanytranscriptionactivationdomains1.yeastGcn4andGal4,2.mammalianglucocorticoidreceptor3.herpesvirusactivatorVP16domains.Transcriptionactivationdomai76Glutamine-richdomainsRichinglutaminetheproportionofglutamineresiduedseemstobemoreimportantthanoverallstructure.ExistsinthegeneraltranscriptionfactorSP1.Glutamine-richdomainsRichin77Proline-richdomainsProline-richcontinuousrunofprolineresiduescanactivatetranscriptionExistsintranscriptionfactorsc-Jun,AP2andOct-2.Proline-richdomainsProline-ri78RepressordomainsRepressionoftranscriptionmayoccurbyindirectinterferencewiththefunctionofanactivator.Thismayoccurby:

1.BlockingtheactivatorDNA-bindingsite2.Formationofanon-DNA-bindingcomplex(e.g.theIdproteinwhichblocksHLHprotein-DNAinteractions,sinceitlacksaDNA-bindingdomain,RepressordomainsRepressionof79

3.MaskingoftheactivationdomainwithoutpreventingDNAbinding(e.g.Gal80maskstheactivationdomainoftheyeasttranscriptionfactorGal4).4.Aspecificdomainoftherepressorisdirectlyresponsibleforinhibitionoftranscription.3.Maskingoftheactivati80Targetsfor

transcriptionalregulation1.chromatinstructure;2.interactionwithTFIIDthroughspecificTAFIIS;3.interactionwithTFIIB;4.interactionormodulationoftheTFIIHcomplexactivityleadingtodifferentialphosphorylationoftheCTDofRNAPolII.Targetsfor

transcriptionalr81Itseemslikelythatdifferentactivationdomainsmayhavedifferenttargets,andalmostanycomponentorstagein

initiation

andtranscriptionelongationcouldbeatargetforregulationresultinginmultistageregulationoftranscriptionItseemslikelythatdifferent82ExamplesoftranscriptionalregulationConstitutivetranscriptionfators:SP1bindstoaGC-richsequencewiththeconsensussequenceGGGCGG.bindingsiteisinthepromoterofmanyhousekeepinggenesItisaconstitutivetranscriptionfactorpresentinallcelltypes.containsthreezincfingermotifsandtwoglutamine-richactivationdomainsinteractingwithTAFII110,thusregulatingthebasaltranscriptioncomplex.Examplesoftranscriptionalre83Hormonalregulation:steroidhormonereceptors

Manytranscriptionfactorsareactivatedbyhormoneswhicharesecretedbyonecelltypeandtransmitasignaltoadifferentcelltype.steroidhormones:lipidsolubleandcandiffusethroughcellmembranestointeractwithtranscriptionfactorscalledsteroidhormonereceptors.Hormonalregulation:Manytran84Intheabsenceofsteroidhormone,thereceptorisboundtoaninhibitor,andlocatedinthecytoplasm.Inthepresenceofsteroidhormone,1.thehormonebindstothereceptorandreleasesthereceptorfromtheinhibitor,2.receptordimerizationandtranslocationtothenucleus.3.receptorinteractsitsspecificDNA-bindingsequence(responseelement)viaitsDNA-bindingdomain,activatingthetargetgene.Intheabsenceofsteroidhorm85relatedimportanthormonereceptors:glucocorticoid,estrogen,retinoicacidandthyroidhormonereceptors.PleasenotedthattheabovemodelisnottrueforallthesehormonereceptorsThyroidhormonereceptor

isaDNA-boundrepressorintheabsenceofhormone,whichconvertedtoatranscriptionalactivatorinthepresenceofthehormone.

relatedimportanthormonerece86分子生物学6真核生物的转录及其调控课件87Regulationbyphosphorylation:STATproteinsForhormonesthatdonotdiffuseintothecell.Thehormonesbindstocell-surfacereceptorsandpassasignaltoproteinswithinthecellthroughsignaltransduction.Signaltransductionofteninvolvesproteinphosphorylation.Example

Interferon-γinducesphosphorylationofatranscriptionfactorcalledSTAT1αthroughactivationoftheintracellularkinasecalledJanusactivatedkinase(JAK).Regulationbyphosphorylation:881.UnphosphorylatedSTAT1αprotein:existsasamonomerinthecellcytoplasmandhasnotranscriptionalactivity.2.PhosphorylatedSTAT1αataspecifictyrosineresidueformsahomodimerwhichmovesintothenucleustoactivatetheexpressionoftargetgeneswhosepromoterregionscontainaconsensusDNA-bindingmotif1.UnphosphorylatedSTAT1αpro89分子生物学6真核生物的转录及其调控课件90Transcriptionelongation:HIVTatHumanimmunodeficiencyvirus(HIV)encodesanactivatorproteincalledTat,whichisrequiredforproductiveHIVgeneexpression.TatbindstoanRNAstem-loopstructurecalledTAR,whichispresentinthe5’-UTRofallHIVRNAsjustaftertheHIVtranscriptionstartsite,toregulatetheleveloftranscriptionelongation.Transcriptionelongation:HIV91IntheabsenceofTat,theHIVtranscriptsterminateprematurelyduetopoorprocessivityoftheRNAPolⅡtranscriptioncomplex.TatbindstoTARononetranscriptinacomplextogetherwithcellularRNA-bindingfactors.Thisprotein-RNAcomplexmayloopbackwardsandinteractwiththenewtranscriptioninitiationcomplexwhichisassembledatthepromoter.IntheabsenceofTat,theHIV92ThisinteractionmayresultintheactivationofthekinaseactivityofTFIIH,leadingtophosphorylationofthecarboxyl-terminaldomain(CTD)ofRNAPolⅡ,makingthepolymeraseaprocessiveenzymetoreadthroughtheHIVtranscriptionunit,leadingtotheproductivesynthesisofHIVproteinsThisinteractionmayresultin93分子生物学6真核生物的转录及其调控课件94Celldetermination:myoDmyoDwasidentifiedasagenetoregulategeneexpressionincelldetermination,commandingcellstoformmuscle.MyoDproteinhasbeenshowntoactivatemuscle-specificgeneexpressiondirectly.OverexpressionofmyoDcanturnfibroblastsintomuscle-likecellswhichexpressmuscle-specificgenesandresemblemyotomes.myoDalsoactivatesexpressionofp21waf1/cip1expression,asmallmoleculeinhibitorofCDKs,causingcellsarrestedattheG1-phaseofthecellcyclewhichischaracteristicofdifferentiatedcells.Celldetermination:myoDmyoDw95Fourgenes,myoD,myogenin,myf5andmrf4havebeenshowntohavetheabilitytoconvertfibroblastsintomuscle.Theencodedproteinsareallmembersofthehelix-loop-helix(HLHfordimerization)transcriptionfactorfamily.TheseproteinsareregulatedbyaninhibitorcalledIdthatlacksaDNA-bindingdomain,butcontainstheHLHdimerizationdomain.IdproteincanbindtoMyoDandrelatedproteins,buttheresultingheterodimerscannotbindDNA,andhencecannotregulatetranscriptionFourgenes,myoD,myogenin,myf596Embryonicdevelopment:homeodomainproteins

ThehomeoboxisaconservedDNAsequencewhichencodesthehelix-turn-helixDNAbindingproteinstructurecalledthehomeodomain.HomeoticgenesofDrosophilaareresponsibleforthecorrectspecificationofbodyparts.Forexample,mutationofoneofthesegenes,Antennapedia,causestheflytoformalegwheretheantennashouldbe.conservedbetweenawiderangeofeukaryotes.importantinmammaliandevelopment.Embryonicdevelopment:Thehome97EssayquestionsGeneralprincipleoftranscription?ThefunctionsofE.coliRNApolymerase’ssubunits?Thedifferencesbetweenlacoperonandtrpoperon?ThedifferencesamongthreeRNApolymerasesineukaryotes?EssayquestionsGeneralprincip98TranscriptioninEukaryotesTranscriptioninEukaryotes99ThethreeRNAPolymerasescharacterizationandfunctionMainFeaturesofeukaryotictranscription1.Themechanismofeukaryotictranscriptionissimilartothatinprokaryotes.2.Alotmoreproteinsareassociatedwiththeeukaryotictranscriptionmachinery,whichresultsinthemuchmorecomplicatedtranscription.ThethreeRNAPolymerasesMain1003.

Threeeukaryoticpolymerasestranscribedifferentsetsofgenes.Theactivitiesofthesepolymerasesaredistinguishedbytheirsensitivitiestothefungaltoxinα-amanitin.4.Inaddition,eukaryoticcellscontainadditionalRNAPolsinmitochondriaandchloroplasts.3.Threeeukaryoticpolymerase101ThreeeukaryoticpolymerasesTypeLocationSubstrateα-amanitinRNAPolINucleoliMostrRNAsgeneInsensitiveRNAPolIINucleoplasmAllprotein-codinggenesandsomesnRNAgenesVerysensitiveRNAPolIIINucleoplasmtRNAs,5SrRNA,U6snRNAandothersmallRNAsModeratelysensitiveThreeeukaryoticpolymerasesTy102α-amanitinThesensitivitiesofthreekindsofRNApolymerasesonα-amanitinα-amanitinThesensitivitiesof103RNApolymerasesubunitsEacheukary