第11章DNA的复制、修复和重组-DNATranscr

DNATranscriptionBasicfeaturesCommontoDNAreplication1)Template,UnwindingandTorsion-relievingarenecessary;2)Proceedonlyinthe5′→3′direction;UncommontoDNAreplication1)Noneedforprimers2)NTPsinsteadofdNTPs;UTPinsteadofdTTP3)Lackingproof-readingactivity(errorrateis1in104or105ntsadded)4)Specificregions(notallDNAsequence)canbetranscribed5)Toaspecificgene,onlyonestrandcanbetranscribedRemembersomenomenclatureconventionsRNA合成与DNA合成的比较：第十三章RNA的代谢（1）催化方向均是5‘－3‘，延伸的机理相同：反应受焦磷酸水解趋动，需要模板。（2）RNA合成不需引物（自身可以独立起始合成），且无外切酶作用（即缺乏核对能力）；DNA复制是一个半保留复制，RNA合成是全保留的（因是单链）。（3）RNA合成起始和终止均受严格的控制，而DNA的终止无特殊的信号。CentralDogmaTranscriptionTranslationReplicationReplicationRetro-transcriptionGeneexpression第一节依赖DNA的RNA合成转录的概念以DNA分子中的某一区段的一条链为模板，在RNA聚合酶的作用下合成一段RNA链。5‘――3‘：CodingstrandP3243‘――5：TemplatestrandCodingstrand,Sensestrand,CrickstrandTemplatestrand,antisensestrand,WatsonstrandTranscriptionTranslation原核生物的RNA聚合酶P3241.亚基的生成2.功能：催化RNA的合成:tRNA，rRNA，mRNA3.抑制剂：利福平rifampicinDNA-DependentRNAPolymerases-RNAP•CommonfeaturesRNAPDNA+NTPs/Mg2+DNA+RNAs+nPPi2nPi•DifferencesbetweenDNAPandRNAP•ProkaryoticRNAP•EukaryoticRNAPs•ViralRNAPsAllRNApolymerasesrequire:1)DNAtemplate:onestrandiscopied2)substrateNTPs(GTP,CTP,UTP,ATP)3)divalentcation(Mg2+)DifferencesBetweenDNAPandRNAP1)RNAPcaninitiatetranscriptiondenovo(i.e.RNAPdoesn’tneedaprimer!)2)RNAPhasnoproofreadingactivity(errorrateis1in104or105ntsadded)3)RNAPincorporatesNTPsinsteadofdNTPs4)RNAPincorporatesUTPinsteadofdTTPRNAPinProkaryotes1)StructureandFunctionAllthreeclassesofRNAsaretranscribedbythesameRNApolymeraseInE.coli,RNAPis465kDcomplex,with2,1,1',1(holoenzyme)Coreenzymeis2,1,1’(cantranscribebutitcan’tfindpromoters)recognizespromotersequencesonDNA2)InhibitorsRifampicin(利福霉素)&Streptolydigin(利链霉素)222’=coreenzymeI’IICOREENZYMESequence-independent,nonspecifictranscriptioninitiation+vegetative(principal)70heatshock(foremergencies)32nitrogenstarvation(foremergencies)60σSUBUNITinterchangeable,promoterrecognitionTheassemblypathwayofthecoreenzyme(thewsubunitmakesthismoreefficient)I’II70RNAPHOLOENZYME-70Promoter-specifictranscriptioninitiationIntheHoloenzyme:'bindsDNAbindsNTPsand'togethermakeuptheactivesitesubunitsappeartobeessentialforassemblyandforactivationofenzymebyregulatoryproteins.TheyalsobindDNA.recognizespromotersequencesonDNARNAPsinEukaryotes•RNApolymerasesI,IIandIIItranscriberRNA,mRNAandtRNAgenes,respectively原核生物的转录过程起始阶段延长阶段终止阶段1、结合2、解链3、引发4、б因子解离5、核心酶移动6、形成3‘，5‘－磷酸二酯键7、因子识别终止信号8、核心酶停止转录9、RNA链释放出来RNA聚合酶（2ββ‘wб）与启动子（promoter）结合，б组别启动子部位（-35启动子部位）。б和β‘起连接作用。结合RNA聚合酶合成RNA的方向为5‘－3‘，所以从转录起始点沿RNA聚合酶运动的方向称为下游（downstream），核苷酸残基编号依次为+2，+3…，而反方向为上游（upstream），核苷酸残基编号为-1，-2…。原核生物的转录过程解开一小段DNA双螺旋，以便产生单链DNA转录模板。解链原核生物的转录过程第一个核苷三磷酸上去（GTP、ATP）（模板）若第一个是C，那么是PPPG（GTP）结合上去。引发б因子的作用：识别启动子部位识别启动部位原核生物的转录过程DetailedTranscriptionalMechanism•Three-stepprocess1)Initiation2)Elongation3)Termination•DNAtranscriptioninprokaryotes•DNAtranscriptionineukaryotes•InvitroDNAtranscriptionProkaryoticDNATranscription•Initiation1)whatispromoter?2)howtodeterminethepromotersequences?-DNaseIfootprinting3)Consensussequences4)Formationoftranscriptionalcomplex•Elongation•TerminationInitiationofTranscription•BindingofRNAPtoTemplateDNA•RNApolymerasehastwobindingsitesforNTPs•InitiationsitepreferstobindsATPandGTP(mostRNAsbeginwithapurineat5'-end)•ElongationsitebindsthesecondincomingNTP•3'-OHoffirstattacksalpha-Pofsecondtoformanewphosphoesterbond(eliminatingPPi)•When6-10unitoligonucleotidehasbeenmade,sigmasubunitdissociates,completinginitiationFindingandbindingthepromoterClosedcomplexformationRNAPbound-40to+20OpencomplexformationRNAPunwindsfrom-10to+2Bindingof1stNTPRequireshighpurine[NTP]AdditionofnextNTPsRequireslower[NTPs]DissociationofsigmaAfterRNAchainis6-10NTPslong4.б因子解离延长阶段5.核心酶移动6.形成3‘，5‘－磷酸二酯键核心酶，特别是其中的β亚基起3‘，5‘－磷酸二酯键的催化作用的核心酶沿模板3‘→5‘移动，RNA链5‘→3‘延长。原核生物的转录过程ChainElongationCorepolymerase-nosigmafactor•Polymeraseisprettyaccurate-onlyabout1errorin10,000bases(notasaccurateasDNAPIII)•EventhiserrorrateisOK,sincemanytranscriptsaremadefromeachgene•Elongationrateis20-50basespersecond-slowerinG/C-richregionsandfasterelsewhere•TopoisomerasesprecedeandfollowpolymerasetorelievesupercoilingScience,vol.281,p424(1998)SpatialOrganizationofTranscriptionElongationComplexinE.coliInteractionsbetweennucleicacidsandthecoreenzymekeepRNAPprocessive7.因子识别终止信号（不衣赖因子）终止阶段8.核心酶不停止转录9.RNA链释放出来Twomechanisms•Rho(ρ)-theterminationfactorprotein•rhoisanATP-dependenthelicase•itmovesalongRNAtranscript,findsthebubble,unwindsitandreleasesRNAchain•Specificsequences-terminationsitesinDNA•invertedrepeat,richinG:C,whichformsastem-loopinRNAtranscript•6-8AsinDNAcodingforUsintranscriptChainTerminationRho-independenttranscriptiontermination(dependsonDNAsequence-NOTaproteinfactor)Stem-loopstructureRho-independenttranscriptiontermination•RNAPpauseswhenitreachesaterminationsite.•Thepausemaygivethehairpinstructuretimetofold•ThefolddisruptsimportantinteractionsbetweentheRNAPanditsRNAproduct•TheU-richRNAcandissociatefromthetemplate•Thecomplexisnowdisr