第11章DNA的复制、修复和重组-DNATranscr课件

DNATranscriptionBasicfeatures

CommontoDNAreplication

1)

Template,UnwindingandTorsion-relievingarenecessary;

2)

Proceedonlyinthe5′→3′direction;UncommontoDNAreplication

1)

Noneedforprimers

2)

NTPsinsteadofdNTPs;UTPinsteadofdTTP

3)

Lackingproof-readingactivity

(errorrateis1in104or105

ntsadded)

4)

Specificregions(notallDNAsequence)canbetranscribed

5)

Toaspecificgene,onlyonestrandcanbetranscribedRemembersomenomenclatureconventionsDNATranscriptionBasicfeature1RNA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。RNA合成与DNA合成的比较：第十三章RNA的代谢（1）2CentralDogmaTranscriptionTranslationReplicationReplicationRetro-transcriptionGeneexpressionCentralDogmaTranscriptionT3第11章DNA的复制、修复和重组-DNATranscr课件4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：

Templatestrand第一节依赖DNA的RNA合成以DNA分子中的某一区5Codingstrand,Sensestrand,CrickstrandTemplatestrand,antisensestrand,WatsonstrandTranscriptionTranslationCodingstrand,Sensestrand,C6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rifampicin原核生物的RNA聚合酶P3241.亚基的生成2.功能：催7DNA-DependentRNAPolymerases

-

RNAPCommonfeatures

RNAPDNA+NTPs/Mg2+

DNA+RNAs+nPPi

2nPiDifferencesbetweenDNAPandRNAPProkaryoticRNAPEukaryoticRNAPsViralRNAPsDNA-DependentRNAPolymerases8AllRNApolymerasesrequire:1)DNAtemplate:onestrandiscopied2)substrateNTPs(GTP,CTP,UTP,ATP)3)divalentcation(Mg2+)DifferencesBetweenDNAPandRNAP1)RNAPcaninitiatetranscriptiondenovo(i.e.RNAPdoesn’tneedaprimer!)2)RNAPhasnoproofreadingactivity(errorrateis1in104or105ntsadded)3)RNAPincorporatesNTPsinsteadofdNTPs4)RNAPincorporatesUTPinsteadofdTTPAllRNApolymerasesrequire:1)9RNAPinProkaryotes

1)StructureandFunction

AllthreeclassesofRNAsaretranscribedbythesameRNApolymerase

InE.coli,RNAPis465kDcomplex,with

2,1,1',1

(holoenzyme)Coreenzymeis2,1,1’(cantranscribebutitcan’tfindpromoters)

recognizespromotersequencesonDNA2)Inhibitors

Rifampicin(利福霉素)&Streptolydigin(利链霉素)RNAPinProkaryotes1)Structu10a a2 a2b a2bb’=coreenzymeaIbb’aIICOREENZYMESequence-independent,nonspecifictranscriptioninitiation+vegetative(principals)s70heatshock(foremergencies)s32nitrogenstarvation(foremergencies)s60σSUBUNITinterchangeable,promoterrecognitionTheassemblypathwayofthecoreenzyme(thewsubunitmakesthismoreefficient)a a2 a2b a2bb’=coreenz11aIbb’aIIs70RNAPHOLOENZYME-s70

Promoter-specifictranscriptioninitiationIntheHoloenzyme:

'bindsDNA

bindsNTPs

and'togethermakeuptheactivesite

subunitsappeartobeessentialforassemblyandforactivationofenzymebyregulatoryproteins.TheyalsobindDNA.

srecognizespromotersequencesonDNAaIbb’aIIs70RNAPHOLOENZYME-s712RNAPsinEukaryotesRNApolymerasesI,IIandIIItranscriberRNA,mRNAandtRNAgenes,respectively

RNAPsinEukaryotesRNApolymer13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14

RNA聚合酶（2ββ‘б）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RNA聚合酶（2ββ‘б）与启动子（promo15

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解开一小段DNA双螺旋，以便产生单链DNA转16

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第一个核苷三磷酸上去（GTP、A17DetailedTranscriptionalMechanismThree-stepprocess

1)Initiation

2)Elongation

3)TerminationDNAtranscriptioninprokaryotesDNAtranscriptionineukaryotesInvitroDNAtranscriptionDetailedTranscriptionalMecha18ProkaryoticDNATranscriptionInitiation

1)whatispromoter?

2)howtodeterminethepromotersequences?-DNaseIfootprinting

3)Consensussequences

4)FormationoftranscriptionalcomplexElongationTerminationProkaryoticDNATranscriptionI19InitiationofTranscription

BindingofRNAPtoTemplateDNARNApolymerasehastwobindingsitesforNTPsInitiationsitepreferstobindsATPandGTP(mostRNAsbeginwithapurineat5'-end)ElongationsitebindsthesecondincomingNTP3'-OHoffirstattacksalpha-Pofsecondtoformanewphosphoesterbond(eliminatingPPi)When6-10unitoligonucleotidehasbeenmade,sigmasubunitdissociates,completing"initiation"

InitiationofTranscriptionBi20FindingandbindingthepromoterClosedcomplexformationRNAPbound-40to+20OpencomplexformationRNAPunwindsfrom-10to+2Bindingof1stNTPRequireshighpurine[NTP]AdditionofnextNTPsRequireslower[NTPs]DissociationofsigmaAfterRNAchainis6-10NTPslongFindingandbindingthepromot214.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4.б因子解离延长阶段5.核心酶移动6.形22ChainElongation

Corepolymerase-nosigma

factorPolymeraseisprettyaccurate-onlyabout1errorin10,000bases(notasaccurateasDNAPIII)EventhiserrorrateisOK,sincemanytranscriptsaremadefromeachgeneElongationrateis20-50basespersecond-slowerinG/C-richregionsandfasterelsewhereTopoisomerasesprecedeandfollowpolymerasetorelievesupercoiling

ChainElongationCorepolymera23Science,vol.281,p424(1998)SpatialOrganizationofTranscriptionElongationComplexinE.coliScience,vol.281,p424(199824InteractionsbetweennucleicacidsandthecoreenzymekeepRNAPprocessiveInteractionsbetweennucleica257.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7.因子识别终止信号（不衣赖因子）终止阶段8.26Twomechanisms

Rho(ρ)-theterminationfactorprotein

rhoisanATP-dependenthelicase

itmovesalongRNAtranscript,findsthe"bubble",unwindsitandreleasesRNAchainSpecificsequences-terminationsitesinDNA

invertedrepeat,richinG:C,whichformsastem-loopinRNAtranscript6-8AsinDNAcodingforUsintranscript

ChainTermination

TwomechanismsChainTerminati27Rho-independenttranscriptiontermination(dependsonDNAsequence-NOTaproteinfactor)Stem-loopstructureRho-independenttranscription28Rho-independenttranscriptiontermination

RNAPpauseswhenitreachesaterminationsite.ThepausemaygivethehairpinstructuretimetofoldThefolddisruptsimportantinteractionsbetweentheRNAPanditsRNAproductTheU-richRNAcandissociatefromthetemplateThecomplexisnowdisruptedandelongationisterminatedRho-independenttranscription29Rho-DependentTranscriptionTermination(dependsonaproteinANDaDNAsequence)G/C-richsiteRNAPslowsdownRhohelicasecatchesupElongatingcomplexisdisruptedRho-DependentTranscriptionTe301、真核有三种RNA聚合酶(P328)原核真核RNA聚合酶只有一种3种利福平受抑制不受抑制转录和蛋白质合成同步不同步第四节真核细胞的转录作用原核真核RNA聚合酶只有一种3种利福平受31如何保证转录的忠实性：(转录差错率为10-5)1、

靠模板链与新合成链之间的碱基配对2、

核心酶对底物有专一性如何保证转录的忠实性：(转录差错率为10-5)1、

靠模板32转录过程的选择性抑制抗菌素放线菌素DP330丫啶利福平-鹅膏覃碱转录过程的选择性抑制33RNAPIIInhibitorMushroomsofthegenusAmanitamakeatoxiccyclicoctapeptidecalledaamanitin

(鹅膏蕈碱）Thismushroomtastesgoodbuteatingitisdeadly!6to24hoursaftereatingitviolentcrampsanddiarrheasetin3rddayseesafalseremissionBy4thor5thdaydeathwilloccurunlessalivertransplantisdoneThesymptomsareduetoinhibitionofRNAPIIandmanifestmainlyinliverRNAPIIInhibitorMushroomsof34Thechemicalstructureofα-amanitinThechemicalstructureofα-am35第11章DNA的复制、修复和重组-DNATranscr课件36RNAPsinEukaryotesAll3arebig,multimericproteins(500-700kD)Allhave2largesubunitswithsequencessimilartoand'inE.coliRNAP,socatalyticsitemaybeconservedAllhavesubunithomologsofainE.coliRNAPHowever,theeukaryoticRNApolymerasedoesnotcontainanysubunitsimilartotheE.coli

σfactor.

ThesefeaturesaresharedbyRNAPsacrossspeciesRNApolymerasesI,IIandIIIhavestructuralfeaturesincommon:

RNAPsinEukaryotesAll3areb37DifferencesTranscriptionBacteriavs.EukaryotesMultiplePolymerases–atleast3typesofRNAPsChromatinandNucleosomesUnabletoinitiatetranscriptionontheirown--RequireTranscriptionFactors(TF，转录因子)UnabletorecognizePromotersontheirownPrimarytranscriptscontainexonsThePromotersarecomplex.Multipleregulatoryproteinscanbindtothepromoter.Cis-actingelements(顺式作用元件)andTrans-actingfactors(反式作用因子).Enhancer,silencer&insulatormRNAsaremostlymonocistronic(单顺反子)Genescontrolledbypositivecontrol-offunlessactivatorsarepresentIneukaryotes,transcriptionandtranslationoccurinseparatecompartments.DifferencesTranscriptionBact38（三）真核生物与原核生物转录的主要区别

1.真核细胞RNApol种类较多，根据它们对α-鹅膏蕈碱的敏感性不同分为RNApolI、II、III（orA、B、C），它们是高度分工的，不同的RNA聚合酶负责合成不同的RNA。

（三）真核生物与原核生物转录的主要区别1.真核细胞RNA392.真核启动子比原核启动子更复杂和更多样性，不同的RNA聚合酶有不同的启动子。

2.真核启动子比原核启动子更复杂和更多样性，不同的403.原核细胞靠RNApol本身可识别启动子，而真核细胞的RNApol无法识别启动子，要靠转录因子(transcriptionfactor,TF）识别启动子，有许多转录因子。转录因子的功能：调节RNA聚合酶的活性，将RNA聚合酶引到启动子位置。3.原核细胞靠RNApol本身可识别启动子，而真核细胞414.真核生物的转录受特定的顺式作用元件（cis-actingelement）的影响，顺式作用元件：真核生物DNA中与转录调控有关的核苷酸序列，包括增强子、沉默子等。沉默子（silencer）：降低转录的速度，沉默子也称抑制子。增强子（enhaucer）：增加转录的速度。4.真核生物的转录受特定的顺式作用元件（cis-acti42顺式作用元件并不能直接发挥作用，要与反式作用因子（trans-actingfactors）相互作用来调控转录，反式作用因子是一些特殊的蛋白质因子。顺式作用元件并不能直接发挥作用，要与反式作用435.原核细胞基因转录的产物大多数为多顺反子mRNA，这是由于原核转录系统中功能相关的基因共享一个启动子，它们在转录时，以一个共同的转录单位进行转录。而真核细胞，每一种蛋白质的基因都有自己独立的启动子，所以真核细胞转录产物是单顺反子mRNA。

5.原核细胞基因转录的产物大多数为多顺反子mRNA，这44原核真核DNAABCP转录mRNAABCDNAPAPBPC转录mRNAABC原核真核DNAABCP转录mRNAABCDNAPAPBPC转456.原核细胞是边转录、边翻译，两个过程几乎是同时进行的，而真核细胞转录和翻译在时间上和空间上都是分开的，转录在细胞核，翻译在细胞质。

7.真核细胞中DNA与组蛋白结合在一起，形成染色质，后者进一步盘曲、折叠形成染色体，其中只有一小部分能转录。8.真核细胞被转录的产物要经过非常复杂的后加工。6.原核细胞是边转录、边翻译，两个过程几乎是同时进7.46RelativepositionsofGTFsandRNAPIIonDNARNAPIIcrabclawsclampovertheDNAneartheinitiationsite.MeltingoftheDNAisassistedbytheTFIIHhelicase(notshown)stimulatedbyTFIIERelativepositionsofGTFsand47Formationofpre-initiationcomplexTBP(TFIID)bindstothepromoterDNA(TATA+Inr)recruitsTFIIBrecruitsRNAPII+TFIIFrecruitTFIIErecruitsandstimulatesTFIIHHelicaseactivityunwindsDNAnearstartsiteKinaseactivityphosphorylatesCTDFormationofpre-initiationco48第11章DNA的复制、修复和重组-DNATranscr课件49AnelongationfactorcalledTFIISstimulateselongationforRNAPII.TFIIFalsohasaroleinelongation.AfterterminationoftranscriptiontheCTDisdephosphorylatedandtheRNAPIIcanre-enterapre-initiationcomplex(PIC)ElongationbyRNAPIIAnelongationfactorcalledTF50POLIIIIFCTDMULTI-STEPMODEL

PRE-INITIATIONCOMPLEXINRTATAUPE-20+1IIDIIAIIBHJEPOLIIIIFCTDMULTI-STEPMODEL

P51MULTI-STEPMODEL

INITIATIONCOMPLEX

PHOSPHORYLATIONOFCTDINRTATAUPE-20+1IIDIIAIIBHJEPOLIIIIFCTDPPPPATPMULTI-STEPMODEL

INITIATIONCO52MULTI-STEPMODEL

PROMOTERCLEARANCEINRTATAUPE-20+1IIDIIAIIBHJEPOLIICTDPPPPRIBONUCLEOTIDESpre-mRNAELONGATIONFACTORSMULTI-STEPMODEL

PROMOTERCLEA53INITIATIONINITIATION54PROMOTERCLEARANCEPROMOTERCLEARANCE55ELONGATIONELONGATION56第五节新生RNA的剪接和修饰一、除去内含子的剪接过程内含子有四种类型第五节新生RNA的剪接和修饰57二、真核生物mRNA进行装饰性加工真核生物转录特点：①转录和翻译在时间及空间上是分割的，保证了精细的调节。②合成的RNA链经过多种加工mRNA5’-cap3’-poly(A)intron去掉exon③核内初级转录产物大部分需要降解核不均－RNAhnRNA20%被降介。二、真核生物mRNA进行装饰性加工①转录和翻译在时间及空间58三、不同RNA剪接方式导致一个基因多种产物四、核糖体RNA和tRNA前体的转录后加工转录出来的RNA称为RNA前体，需要进行加工原始转录产物→成熟RNA分子此过程称“转录后加工”。三、不同RNA剪接方式导致一个基因多种产物59

（四）转录产物的“加工”（四）转录产物的“加工”601、核糖体RNA前体的转录后加工2、tRNA前体的加工3、真核细胞mRNA前体的加工：真核细胞mRNA的结构特点P266返回1、核糖体RNA前体的转录后加工返回61①除去前体5‘和3‘端多余的核苷酸；2、tRNA前体的加工②有些tRNA还需要添加3‘端的－CCA三核苷酸顺序；③有些碱需修饰，包括甲基化、脱氨、还原作用等。转录产物的“加工”返回①除去前体5‘和3‘端多余的核苷酸；2、tRNA前体的加工②62内含子（intron）：3、真核细胞mRNA前体的加工基因的插入顺序为不能编码时DNA顺序。外显子（exon）：基因内被内因子分隔的编码片段或编码的DNA序列。转录产物的“加工”返回内含子（intron）：3、真核细胞mRNA前体的加工基因的63

mRNA前体的转录后加工，包括了对5‘端和3‘端（首、尾部）的修饰以及对中间的部分进行剪接（splicing）。转录产物的“加工”返回mRNA前体的转录后加工，包括了对5‘端和3‘端（首64(1)首、尾的修饰在核内经首、尾两部分修饰，在专一的酶促作用下，5‘端形成特殊的帽子结构。P269图大多数mRNA的3‘未端有polyA以ATP为底物，在RNA末端腺苷酸转移酶的作用下，在3‘端逐个添加多聚腺苷酸尾巴。（2）mRNA的剪接P268大多数真核生物基因都是断裂基因，断裂基因的转录产物需通过拼接转录产物的“加工”返回(1)首、尾的修饰（2）mRNA的剪接P268转录产物的“65Post-transcriptional

ProcessingofRNAsDefinitionPrimaryRNAsMatureRNAs

ProcessingofPre-mRNAs

1)InProkaryotes:Rare

2)InEukaryotes:CommonProcessingofPre-rRNAsandPre-tRNAs

1)InProkaryotes

2)InEukaryotesPost-transcriptional

Processi66Post-transcriptionalProcessingofmRNAinEukaryotesProcessingtypes

1)

5’end=capping（带帽）

2)

3’end=tailing（加尾）3)

Internal=splicing（剪接）4)

Internalmethylation（内部甲基化）5)

Editing（编辑）MechanismPost-transcriptionalProcessin67第11章DNA的复制、修复和重组-DNATranscr课件68CappingandMethylation

Strutureandtypes

0,IandIItypeCappingreaction:co-transcriptional

1)EnzymesPhosphohydrolase,guanylyltransferaseandmethyltransferases

2)StepsFunctionsCappingandMethylationStrutu69CAP0CAP1CAP2AdditionalmethylationsarepossibleCAP0CAP1CAP2Additional70WhyhaveaCap?TheCAPstructurepromotesstabilityofthemRNA(preventsdegradationby5’exonucleases)

TheCAPstructurepromotestranslationofthemRNA

AribosomalproteinsubunitinteractswiththeCAPandrecruitsmRNAtotheribosome

WhyhaveaCap?TheCAPstructu71WhyareonlymRNAsCapped?TheCAPstructureisonlyaddedtotranscriptssynthesizedbyRNAPII(i.e.mRNAtranscripts)ThisisbecausethecappingenzymesbindtoapartofRNAPIIthatisuniquetothatenzyme(notfoundinRNAPIorRNAPIII)CappingenzymesbindtotheCTDofRNAPIIWhyareonlymRNAsCapped?The72WhyareonlymRNAsCapped?TheybindonlytotheelongatingformoftheCTD(I.e.thephosphorylatedCTD)AssoonasthetranscriptemergesfromRNAPIIitisincontactwiththecappingenyzmesWhyareonlymRNAsCapped?73第11章DNA的复制、修复和重组-DNATranscr课件743'-Polyadenylation

Apoly(A)tailisaddedtothe3’endofthetranscriptin2steps…1)

cleavage:theRNAiscut10-30nucleotidesdownstreamofaspecificsequenceinthe3’UTR2)

additionofA’s(100-200areadded)togenerateapoly(A)tail

CodingsequenceofthegeneTATAbox+125-30bpmRNATranslatedregion5’UTR3’UTRATGStop3'-PolyadenylationApoly(A)t75mRNA5’CAPAAUAAACPSF1)CPSF=“cleavage/polyadenylationspecificityfactor”(3subunits)recognizestheAAUAAAsequenceintheRNA3’UTRandbindstoit2)RecruitsCFIandCFII=“cleavagefactors”3)RecruitsPAP=“poly(A)polymerase”CFICFIIPAPAribonucleoproteincomplexmRNA5’CAPAAUAAACPSF1)CPSF=76mRNA5’CAPAAUAAACPSF

TheRNAiscleaved10-30nucleotidesdownstreamoftheAAUAAAbyCFI/II

Thisgeneratesa3’OHtowhichPAPaddsAresidues.Itdoesn’tcopyfromatemplate-justneedsa3’OHofanRNA

Thepoly(A)tailisboundbypoly(A)-bindingprotein

CFICFIIPAPAAUAAAAAAAAAAAAAAAA100-200mRNA5’CAPAAUAAACPSFTheRNAi77WhyhaveaPoly(A)tail?Thepoly(A)tailpromotesstabilityofthemRNA(preventsdegradationby3’exonucleases)

Thepoly(A)tailpromotestranslationofthemRNA

AribosomalproteinsubunitthatinteractswiththeCAPstructureisstimulatedinCAPbindingbypoly(A)bindingprotein

ThereforetheCAPandthepoly(A)tailworksynergisticallytorecruitmRNAtotheribosomeCreatestopcodonAlternativetailingprovidesthepossibility-onegene,twoormoreproteinsWhyhaveaPoly(A)tail?Thepo78第11章DNA的复制、修复和重组-DNATranscr课件79EukaryoticGenesareSplit

Discover:RichardJ.RobertsandPhillipA.Sharp(TheNobelPrizelaureateinPhysiologyorMedicine1993)Introns

intervenebetweenexons

Examples:actingenehas309-bpintronseparatesfirstthreeaminoacidsandtheother350orsoButchickenpro-alpha-2collagengeneis40-kbplong,with51exonsofonly5kbptotal.Theexonsrangeinsizefrom45to249basesMechanismbywhichintronsareexcisedandexonsaresplicedtogetheriscomplexandmustbeprecise

EukaryoticGenesareSplitDis80DenatureHybridizewithmaturemRNAAndvisualizeunderEMR-looptechniquesDNAtemplatestrandMaturemRNADenatureHybridizewithmature81第11章DNA的复制、修复和重组-DNATranscr课件82第11章DNA的复制、修复和重组-DNATranscr课件83SplicingofPre-mRNA

Splicingsignals-“internalcause”snRNPs-“externalcause”Splicingreaction-twoTransesterificationreactionsAssemblyofspliceosomesSplicingofPre-mRNASplicing84Sequencerequirementsforsplicing

G3’Exon5’ExonAGGUAGIntron18-40nucleotides3.BranchpointYNYRAYY=pyrimidineR=purineN=anything1.5’splicesite2.3’splicesiteSequencerequirementsforspli85SplicingoccursthroughtwoTransesterificationreactionsPO-OO-O-XYRNAChain+R-OHPO-OO-O-RYX-OH+InatransesterificationreactionaphosphodiesterbondistransferredtoadifferenthydroxylgroupThereisnohydrolysisandnoenergylossSplicingoccursthroughtwoTr86第11章DNA的复制、修复和重组-DNATranscr课件87第11章DNA的复制、修复和重组-DNATranscr课件88Alternativesplicing&Trans-splicing

Alternativesplicing

SplicingispreciseBUT...

Individualpre-mRNAmaybesplicedtomorethanonekindofmRNAbyremovingdifferentcombinationsofintrons/exons

AlternativeformscanberegulatedSexdeterminationinDrosophilaTrans-splicingDefinition-5'and3'junctionsondifferentRNAmoleculesRelativelyrare-otherwiseOccursnaturallyintrypanosomesandinC.elegans-maybeelsewheretoo.Alternativesplicing&Trans-s89AlternativesplicingcangeneratemultipleproteinsfromasinglegeneAlternativesplicingcangener90第11章DNA的复制、修复和重组-DNATranscr课件91CappingTailingCappingTailing92Post-transcriptional

processingofpre-rRNAInkaryotes

Cleavage,trimmingandmodificationIneukaryotes

1)

Cleavage,trimmingandmodification2)

Splicing

(SomeeukaryotessuchasTetrahymena-四膜虫)Post-transcriptional

processi93第11章DNA的复制、修复和重组-DNATranscr课件94第11章DNA的复制、修复和重组-DNATranscr课件95Self-splicingofPre-rRNADiscoveryT.Cech(1989NobelPrizeLaureateinChemistry)found26SrRNAhasone400bintroninsomeTetrahymenastrainsin1981:ThisIntroncanSelf-Splice;requiresionsandaG-nuc(GTP,GDP,GMP,GR)anddoesn’trequireanyproteinsL-19RNAenzymaticactivities

1)NucleotidyltransferaseorRNApolymerase

2)Endoribonuclease;RNAligase;Phosphatase

Self-splicingofPre-rRNADisco96IntronExon1Exon2IntronExon1Exon297第11章DNA的复制、修复和重组-DNATranscr课件98第11章DNA的复制、修复和重组-DNATranscr课件99Post-transcriptional

processingofpre-tRNAInProkaryotes

1)

Cleavageandtrimming

2)

Modification

3)

AdditionofCCA

(ifnecessary)Ineukaryotes

1)

Cleavageandtrimming

2)

Modification

3)

AdditionofCCA

4)

Splicing

(someeukaryotes)Post-transcriptional

processi100第11章DNA的复制、修复和重组-DNATranscr课件101RNaseP-AtrueribozymeDiscoverer-SidneyAltman

(1989NobelPrizeLaureateinChemistry)StructureandFunction

1)

Anendonuclease–involvedin5’-endprocessingofpre-tRNA

2)

E.coliRNaseP:14-kDapolypeptide+a377-nucleotideRNA(M1RNA).

3)

Athigh

Mg2+

concentrations,isolatedM1RNArecognizesandcleavesE.colipre-tRNAs.TheRNasePpolypeptideincreasestherateofcleavagebyM1RNA,allowingittoproceedatphysiological

Mg2+

concentrations.

RNaseP-AtrueribozymeDisco102第11章DNA的复制、修复和重组-DNATranscr课件103第11章DNA的复制、修复和重组-DNATranscr课件104RNAsthatfunctionasenzymesTypes

1)

RNaseP

2)

GroupIintrons

(Foundinpre-rRNAsfromsomesingle-celledorganisms,inmitochondrialandchloroplastpre-rRNAs,inseveralpre-mRNAsfromcertainE.colibacteriophages,andinsomebacterialtRNAprimarytranscripts

)3)

GroupIIintrons

(Foundincertainmitochondrialandchloroplastpre-mRNAs)4)

23SrRNA:peptidebondformation

5)

Hammerheadribozymes

(someplantviruses)6)

snRNAsinvolvedinsplicingFeaturesSignificance–“RNAworld”RNAsthatfunctionasenzymesT105第11章DNA的复制、修复和重组-DNATranscr课件106Whowasproducedfirst,DNAor

Protein？RNA，RNA，RNARNA，RNA，RNA，RNARNA，RNA，RNARNA，RNA，RNARNAworldDNAProteinWhowasproducedfirst,DNAor107Retro-transcriptionDiscovery1960's:HowardTeminknewthatretrovirusgenomeswerecomposedofRNAandobservedthatreplicationwasinhibitedbyactinomycinDthereforeheproposedtheconceptofreversetranscription(NobelprizeawardedtoBaltimoreandTemin,1975).

1970:TeminandDavidBaltimore(separately)discovertheRNA-directedDNApolymerase-"reversetrascriptase"

Retro-virusesRetro-transcriptaseThreeenzymaticactivities:RDNAP,DNAPandRNaseHcDNARetro-transcriptionDiscovery108Seeyounexttime!Seeyounexttime!109演讲完毕，谢谢观看！演讲完毕，谢谢观看！110DNATranscriptionBasicfeatures

CommontoDNAreplication

1)

Template,UnwindingandTorsion-relievingarenecessary;

2)

Proceedonlyinthe5′→3′direction;UncommontoDNAreplication

1)

Noneedforprimers

2)

NTPsinsteadofdNTPs;UTPinsteadofdTTP

3)

Lackingproof-readingactivity

(errorrateis1in104or105

ntsadded)

4)

Specificregions(notallDNAsequence)canbetranscribed

5)

Toaspecificgene,onlyonestrandcanbetranscribedRemembersomenomenclatureconventionsDNATranscriptionBasicfeature111RNA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。RNA合成与DNA合成的比较：第十三章RNA的代谢（1）112CentralDogmaTranscriptionTranslationReplicationReplicationRetro-transcriptionGeneexpressionCentralDogmaTranscriptionT113第11章DNA的复制、修复和重组-DNATranscr课件114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：

Templatestrand第一节依赖DNA的RNA合成以DNA分子中的某一区115Codingstrand,Sensestrand,CrickstrandTemplatestrand,antisensestrand,WatsonstrandTranscriptionTranslationCodingstrand,Sensestrand,C116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rifampicin原核生物的RNA聚合酶P3241.亚基的生成2.功能：催117DNA-DependentRNAPolymerases

-

RNAPCommonfeatures

RNAPDNA+NTPs/Mg2+

DNA+RNAs+nPPi

2nPiDifferencesbetweenDNAPandRNAPProkaryoticRNAPEukaryoticRNAPsViralRNAPsDNA-DependentRNAPolymerases118AllRNApolymerasesrequire:1)DNAtemplate:onestrandiscopied2)substrateNTPs(GTP,CTP,UTP,ATP)3)divalentcation(Mg2+)DifferencesBetweenDNAPandRNAP1)RNAPcaninitiatetranscriptiondenovo(i.e.RNAPdoesn’tneedaprimer!)2)RNAPhasnoproofreadingactivity(errorrateis1in104or105ntsadded)3)RNAPincorporatesNTPsinsteadofdNTPs4)RNAPincorporatesUTPinsteadofdTTPAllRNApolymerasesrequire:1)119RNAPinProkaryotes

1)StructureandFunction

AllthreeclassesofRNAsaretranscribedbythesameRNApolymerase

InE.coli,RNAPis465kDcomplex,with

2,1,1',1

(holoenzyme)Coreenzymeis2,1,1’(cantranscribebutitcan’tfindpromoters)

recognizespromotersequencesonDNA2)Inhibitors

Rifampicin(利福霉素)&Streptolydigin(利链霉素)RNAPinProkaryotes1)Structu120a a2 a2b a2bb’=coreenzymeaIbb’aIICOREENZYMESequence-independent,nonspecifictranscriptioninitiation+vegetative(principals)s70heatshock(foremergencies)s32nitrogenstarvation(foremergencies)s60σSUBUNITinterchangeable,promoterrecognitionTheassemblypathwayofthecoreenzyme(thewsubunitmakesthismoreefficient)a a2 a2b a2bb’=coreenz121aIbb’aIIs70RNAPHOLOENZYME-s70

Promoter-specifictranscriptioninitiationIntheHoloenzyme:

'bindsDNA

bindsNTPs

and'togethermakeuptheactivesite

subunitsappeartobeessentialforassemblyandforactivationofenzymebyregulatoryproteins.TheyalsobindDNA.

srecognizespromotersequencesonDNAaIbb’aIIs70RNAPHOLOENZYME-s7122RNAPsinEukaryotesRNApolymerasesI,IIandIIItranscriberRNA,mRNAandtRNAgenes,respectively

RNAPsinEukaryotesRNApolymer123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124

RNA聚合酶（2ββ‘б）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RNA聚合酶（2ββ‘б）与启动子（promo125

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解开一小段DNA双螺旋，以便产生单链DNA转126

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第一个核苷三磷酸上去（GTP、A127DetailedTranscriptionalMechanismThree-stepprocess

1)Initiation

2)Elongation

3)TerminationDNAtranscriptioninprokaryotesDNAtranscriptionineukaryotesInvitroDNAtranscriptionDetailedTranscriptionalMecha128ProkaryoticDNATranscriptionInitiation

1)whatispromoter?

2)howtodetermineth