武汉大学分子生物学讲义英文

WelcomeEachofYoutoMyMolecularBiologyClass2MolecularBiologyoftheGene,5/E---Watsonetal.(2004)PartI:ChemistryandGeneticsPartII:MaintenanceoftheGenomePartIII:ExpressionoftheGenomePartIV:RegulationPartV:MethodsTherevisedcentraldogma2008RNAprocessingGeneregulationCh6:ThestructuresofDNAandRNACh8:ThereplicationofDNA基因组的保持基因组的表达4Ch12:MechanismsoftranscriptionCh13:RNAsplicingCh14:TranslationCh15:ThegeneticcodePartIII:ExpressionoftheGenomeChapter12:MechanismsofTranscription1.RNApolymeraseandthetranscriptioncycle2.Thetranscriptioncycleinbacteria3.Transcriptionineukaryotes4.TranscriptionbyRNApolymeraseIandIIIMolecularBiologyCourseTranscriptionvsreplication6TranscriptionischemicallyandenzymaticallyverysimilartoDNAreplication.7Someimportantdifferences:1.RNAismadeofribonucleotides2.RNApolymerasecatalyzesthereaction,whichdoesnotrequireaprimer(denovosynthesis)3.TheRNAproductdoesnotremainbase-pairedtothetemplateDNAstrand4.Lessaccurate(errorrate:10-4)85.Transcriptionselectivelycopiesonlycertainpartsofthegenomeandmakesonetoseveralhundred,oreventhousand,copiesofanygivensectionofthegenome.(Replication?)9Fig12-1TranscriptionofDNAintoRNATranscriptionbubble10Topic1:RNAPolymeraseandTheTranscriptionCycleCHAPTER12:MechanismsofTranscriptionSeetheinteractiveanimation11RNApolymerasescomeindifferentforms,butsharemanyfeaturesRNApolymerasesperformsessentiallythesamereactioninallcellsBacteriahaveonlyasingleRNApolymeraseswhileineukaryoticcellstherearethree:RNAPolI,IIandIIIRNApolymeraseandthetranscriptioncycle12RNAPolIIisthefocusofeukaryotictranscription,becauseitisthemoststudiedpolymerase,andisalsoresponsiblefortranscribingmostgenes-indeed,essentiallyallprotein-encodinggenesRNAPolItranscribethelargeribosomalRNAprecursorgeneRNAPolIIItranscribetRNAgene,somesmallnuclearRNAgenesandthe5SrRNAgenes13Table12-1:ThesubunitsofRNApolymerases14ThebacterialRNApolymeraseThecoreenzymealonesynthesizesRNAaabb’w15aab’wRPB3RPB11RPB2RPB1RPB6Fig12-2RNAPComparisonThesamecolorindicatethehomologousofthetwoenzymesprokaryoticeukaryoticb16“Crabclaw”shapeofRNAP(TheshapeofDNApolis__)Activecentercleft17TherearevariouschannelsallowingDNA,RNAandribonucleotides(rNTPs)intoandoutoftheenzyme’sactivecentercleft18TranscriptionbyRNApolymeraseproceedsinaseriesofstepsInitiationElongationTerminationRNApolymeraseandthetranscriptioncycle19InitiationPromoter:theDNAsequencethatinitiallybindstheRNApolymeraseThestructureofpromoter-polymerasecomplexundergoesstructuralchangestoproceedtranscriptionDNAatthetranscriptionsiteunwindsanda“bubble”formsDirectionofRNAsynthesisoccursina5’-3’direction(3’-endgrowing)20Transcriptioninitiationinvolves3definedsteps1.Formingclosedcomplex2.Formingopencomplex3.Promoterescape:stableternarycomplexRNApolymeraseandthetranscriptioncycle21TheinitialbindingofpolymerasetoapromoterDNAremainsdoublestrandedTheenzymeisboundtoonefaceofthehelixClosedcomplex22OpencomplextheDNAstrandseparateoveradistanceof~14bp(-11to+3)aroundthestartsite(+1site)transcriptionbubbleforms23StableternarycomplexTheenzymeescapesfromthepromoterThetransitiontotheelongationphaseStableternarycomplex=DNA+RNA+enzyme24Fig12-3-initiationBinding(closedcomplex)Promoter“melting”(opencomplex)Initialtranscription25ElongationOncetheRNApolymerasehassynthesizedashortstretchofRNA(~10nt),transcriptionshiftsintotheelongationphase.Thistransitionrequiresfurtherconformationalchangeinpolymerasethatleadsittogripthetemplatemorefirmly.Functions:synthesisRNA,unwindstheDNAinfront,re-annealsitbehind,dissociatesthegrowingRNAchain26TerminationAfterthepolymerasetranscribesthelengthofthegene(orgenes),itwillstopandreleasetheRNAtranscript.Insomecells,terminationoccursatthespecificandwell-definedDNAsequencescalledterminators.Somecellslacksuchterminationsequences.27Fig12-3-ElongationandterminationTerminationElongation28Topic2ThetranscriptioncycleinbacteriaCHAPTER12:MechanismsofTranscription292-1Bacterialpromotersvaryinstrengthandsequences,buthavecertaindefiningfeaturesThetranscriptioncycleinbacteria30Figure12-4,Holoenzyme=factor+coreenzymeIncell,RNApolymeraseinitiatestranscriptiononlyatpromoters.Whoconfersthepolymerasebindingspecificity?31ThepredominantfactorinE.coliis70.Promoterrecognizedby70containstwoconservedsequences(-35and–10regions/elements)separatedbyanon-specificstretchof17-19nt.Position+1isthetranscriptionstartsite.PromotersrecognizedbyE.colifactor32Fig12-5a:bacterialpromoterThedistanceisconserved1.70promoterscontainrecognizable–35and–10regions,butthesequencesarenotidentical.2.Comparisonofmanydifferentpromotersderivestheconsensussequencesreflectingpreferred–10and–35regions33BOX12-1Figure1Consensussequenceofthe-35and-10region343.Promoterswithsequencesclosertotheconsensusaregenerally“stronger”thanthosematchlesswell.(Whatdoes“stronger”mean?)4.Thestrengthofthepromoterdescribeshowmanytranscriptsitinitiatesinagiventime.35Fig12-5bbacterialpromoterConfersadditionalspecificityUP-elementisanadditionalDNAelementsthatincreasespolymerasebindingbyprovidingtheadditionalinteractionsiteforRNApolymerase36Fig12