第11章DNA的復(fù)制、修復(fù)和重組-DNATranscr課件

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合成與DNA合成的比較：第十三章RNA的代謝

（1）催化方向均是5‘－3‘，延伸的機(jī)理相同：反應(yīng)受焦磷酸水解趨動(dòng)，需要模板。（2）RNA合成不需引物（自身可以獨(dú)立起始合成），且無(wú)外切酶作用（即缺乏核對(duì)能力）；DNA復(fù)制是一個(gè)半保留復(fù)制，RNA合成是全保留的（因是單鏈）。（3）RNA合成起始和終止均受嚴(yán)格的控制，而DNA的終止無(wú)特殊的信號(hào)。RNA合成與DNA合成的比較：第十三章RNA的代謝（1）2CentralDogmaTranscriptionTranslationReplicationReplicationRetro-transcriptionGeneexpressionCentralDogmaTranscriptionT3第11章DNA的復(fù)制、修復(fù)和重組-DNATranscr課件4第一節(jié)依賴DNA的RNA合成轉(zhuǎn)錄的概念

以DNA分子中的某一區(qū)段的一條鏈為模板，在RNA聚合酶的作用下合成一段RNA鏈。5‘――3‘：CodingstrandP3243‘――5：

Templatestrand第一節(jié)依賴DNA的RNA合成以DNA分子中的某一區(qū)5Codingstrand,Sensestrand,CrickstrandTemplatestrand,antisensestrand,WatsonstrandTranscriptionTranslationCodingstrand,Sensestrand,C6原核生物的RNA聚合酶P3241.亞基的生成2.功能：催化RNA的合成:tRNA，rRNA，mRNA3.抑制劑：利福平rifampicin原核生物的RNA聚合酶P3241.亞基的生成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(利鏈霉素)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原核生物的轉(zhuǎn)錄過程

起始階段延長(zhǎng)階段終止階段1、結(jié)合2、解鏈3、引發(fā)4、б因子解離5、核心酶移動(dòng)6、形成3‘，5‘－磷酸二酯鍵7、因子識(shí)別終止信號(hào)8、核心酶停止轉(zhuǎn)錄9、RNA鏈釋放出來(lái)原核生物的轉(zhuǎn)錄過程 起始階段延長(zhǎng)階段終止階段1、結(jié)合2、解鏈14

RNA聚合酶（2ββ‘б）與啟動(dòng)子（promoter）結(jié)合，б組別啟動(dòng)子部位（-35啟動(dòng)子部位）。б和β‘起連接作用。結(jié)合

RNA聚合酶合成RNA的方向?yàn)?‘－3‘，所以從轉(zhuǎn)錄起始點(diǎn)沿RNA聚合酶運(yùn)動(dòng)的方向稱為下游（downstream），核苷酸殘基編號(hào)依次為+2，+3…，而反方向?yàn)樯嫌危╱pstream），核苷酸殘基編號(hào)為-1，-2…。原核生物的轉(zhuǎn)錄過程RNA聚合酶（2ββ‘б）與啟動(dòng)子（promo15

解開一小段DNA雙螺旋，以便產(chǎn)生單鏈DNA轉(zhuǎn)錄模板。解鏈原核生物的轉(zhuǎn)錄過程解開一小段DNA雙螺旋，以便產(chǎn)生單鏈DNA轉(zhuǎn)16

第一個(gè)核苷三磷酸上去（GTP、ATP）（模板）若第一個(gè)是C，那么是PPPG（GTP）結(jié)合上去。

引發(fā)б因子的作用：識(shí)別啟動(dòng)子部位識(shí)別啟動(dòng)部位原核生物的轉(zhuǎn)錄過程第一個(gè)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因子解離延長(zhǎng)階段5.核心酶移動(dòng)

6.形成3‘，5‘－磷酸二酯鍵

核心酶，特別是其中的β亞基起3‘，5‘－磷酸二酯鍵的催化作用的核心酶沿模板3‘→5‘移動(dòng)，RNA鏈5‘→3‘延長(zhǎng)。原核生物的轉(zhuǎn)錄過程4.б因子解離延長(zhǎng)階段5.核心酶移動(dòng)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.因子識(shí)別終止信號(hào)（不衣賴因子）終止階段8.核心酶不停止轉(zhuǎn)錄9.RNA鏈釋放出來(lái)7.因子識(shí)別終止信號(hào)（不衣賴因子）終止階段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、真核有三種RNA聚合酶(P328)原核真核RNA聚合酶只有一種3種利福平受抑制不受抑制轉(zhuǎn)錄和蛋白質(zhì)合成同步不同步第四節(jié)真核細(xì)胞的轉(zhuǎn)錄作用原核真核RNA聚合酶只有一種3種利福平受31如何保證轉(zhuǎn)錄的忠實(shí)性：(轉(zhuǎn)錄差錯(cuò)率為10-5)1、

靠模板鏈與新合成鏈之間的堿基配對(duì)2、

核心酶對(duì)底物有專一性如何保證轉(zhuǎn)錄的忠實(shí)性：(轉(zhuǎn)錄差錯(cuò)率為10-5)1、

靠模板32轉(zhuǎn)錄過程的選擇性抑制抗菌素放線菌素DP330丫啶利福平-鵝膏覃堿轉(zhuǎn)錄過程的選擇性抑制33RNAPIIInhibitorMushroomsofthegenusAmanitamakeatoxiccyclicoctapeptidecalledaamanitin

(鵝膏蕈堿）Thismushroomtastesgoodbuteatingitisdeadly!6to24hoursaftereatingitviolentcrampsanddiarrheasetin3rddayseesafalseremissionBy4thor5thdaydeathwilloccurunlessalivertransplantisdoneThesymptomsareduetoinhibitionofRNAPIIandmanifestmainlyinliverRNAPIIInhibitorMushroomsof34Thechemicalstructureofα-amanitinThechemicalstructureofα-am35第11章DNA的復(fù)制、修復(fù)和重組-DNATranscr課件36RNAPsinEukaryotesAll3arebig,multimericproteins(500-700kD)Allhave2largesubunitswithsequencessimilartoand'inE.coliRNAP,socatalyticsitemaybeconservedAllhavesubunithomologsofainE.coliRNAPHowever,theeukaryoticRNApolymerasedoesnotcontainanysubunitsimilartotheE.coli

σfactor.

ThesefeaturesaresharedbyRNAPsacrossspeciesRNApolymerasesI,IIandIIIhavestructuralfeaturesincommon:

RNAPsinEukaryotesAll3areb37DifferencesTranscriptionBacteriavs.EukaryotesMultiplePolymerases–atleast3typesofRNAPsChromatinandNucleosomesUnabletoinitiatetranscriptionontheirown--RequireTranscriptionFactors(TF，轉(zhuǎn)錄因子)UnabletorecognizePromotersontheirownPrimarytranscriptscontainexonsThePromotersarecomplex.Multipleregulatoryproteinscanbindtothepromoter.Cis-actingelements(順式作用元件)andTrans-actingfactors(反式作用因子).Enhancer,silencer&insulatormRNAsaremostlymonocistronic(單順反子)Genescontrolledbypositivecontrol-offunlessactivatorsarepresentIneukaryotes,transcriptionandtranslationoccurinseparatecompartments.DifferencesTranscriptionBact38（三）真核生物與原核生物轉(zhuǎn)錄的主要區(qū)別

1.真核細(xì)胞RNApol種類較多，根據(jù)它們對(duì)α-鵝膏蕈堿的敏感性不同分為RNApolI、II、III（orA、B、C），它們是高度分工的，不同的RNA聚合酶負(fù)責(zé)合成不同的RNA。

（三）真核生物與原核生物轉(zhuǎn)錄的主要區(qū)別1.真核細(xì)胞RNA392.真核啟動(dòng)子比原核啟動(dòng)子更復(fù)雜和更多樣性，不同的RNA聚合酶有不同的啟動(dòng)子。

2.真核啟動(dòng)子比原核啟動(dòng)子更復(fù)雜和更多樣性，不同的403.原核細(xì)胞靠RNApol本身可識(shí)別啟動(dòng)子，而真核細(xì)胞的RNApol無(wú)法識(shí)別啟動(dòng)子，要靠轉(zhuǎn)錄因子(transcriptionfactor,TF）識(shí)別啟動(dòng)子，有許多轉(zhuǎn)錄因子。轉(zhuǎn)錄因子的功能：調(diào)節(jié)RNA聚合酶的活性，將RNA聚合酶引到啟動(dòng)子位置。3.原核細(xì)胞靠RNApol本身可識(shí)別啟動(dòng)子，而真核細(xì)胞414.真核生物的轉(zhuǎn)錄受特定的順式作用元件（cis-actingelement）的影響，順式作用元件：真核生物DNA中與轉(zhuǎn)錄調(diào)控有關(guān)的核苷酸序列，包括增強(qiáng)子、沉默子等。沉默子（silencer）：降低轉(zhuǎn)錄的速度，沉默子也稱抑制子。增強(qiáng)子（enhaucer）：增加轉(zhuǎn)錄的速度。4.真核生物的轉(zhuǎn)錄受特定的順式作用元件（cis-acti42順式作用元件并不能直接發(fā)揮作用，要與反式作用因子（trans-actingfactors）相互作用來(lái)調(diào)控轉(zhuǎn)錄，反式作用因子是一些特殊的蛋白質(zhì)因子。順式作用元件并不能直接發(fā)揮作用，要與反式作用435.原核細(xì)胞基因轉(zhuǎn)錄的產(chǎn)物大多數(shù)為多順反子mRNA，這是由于原核轉(zhuǎn)錄系統(tǒng)中功能相關(guān)的基因共享一個(gè)啟動(dòng)子，它們?cè)谵D(zhuǎn)錄時(shí)，以一個(gè)共同的轉(zhuǎn)錄單位進(jìn)行轉(zhuǎn)錄。而真核細(xì)胞，每一種蛋白質(zhì)的基因都有自己獨(dú)立的啟動(dòng)子，所以真核細(xì)胞轉(zhuǎn)錄產(chǎn)物是單順反子mRNA。

5.原核細(xì)胞基因轉(zhuǎn)錄的產(chǎn)物大多數(shù)為多順反子mRNA，這44原核真核DNAABCP轉(zhuǎn)錄mRNAABCDNAPAPBPC轉(zhuǎn)錄mRNAABC原核真核DNAABCP轉(zhuǎn)錄mRNAABCDNAPAPBPC轉(zhuǎn)456.原核細(xì)胞是邊轉(zhuǎn)錄、邊翻譯，兩個(gè)過程幾乎是同時(shí)進(jìn)行的，而真核細(xì)胞轉(zhuǎn)錄和翻譯在時(shí)間上和空間上都是分開的，轉(zhuǎn)錄在細(xì)胞核，翻譯在細(xì)胞質(zhì)。

7.真核細(xì)胞中DNA與組蛋白結(jié)合在一起，形成染色質(zhì)，后者進(jìn)一步盤曲、折疊形成染色體，其中只有一小部分能轉(zhuǎn)錄。8.真核細(xì)胞被轉(zhuǎn)錄的產(chǎn)物要經(jīng)過非常復(fù)雜的后加工。6.原核細(xì)胞是邊轉(zhuǎn)錄、邊翻譯，兩個(gè)過程幾乎是同時(shí)進(jìn)7.46RelativepositionsofGTFsandRNAPIIonDNARNAPIIcrabclawsclampovertheDNAneartheinitiationsite.MeltingoftheDNAisassistedbytheTFIIHhelicase(notshown)stimulatedbyTFIIERelativepositionsofGTFsand47Formationofpre-initiationcomplexTBP(TFIID)bindstothepromoterDNA(TATA+Inr)recruitsTFIIBrecruitsRNAPII+TFIIFrecruitTFIIErecruitsandstimulatesTFIIHHelicaseactivityunwindsDNAnearstartsiteKinaseactivityphosphorylatesCTDFormationofpre-initiationco48第11章DNA的復(fù)制、修復(fù)和重組-DNATranscr課件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第五節(jié)新生RNA的剪接和修飾一、除去內(nèi)含子的剪接過程內(nèi)含子有四種類型第五節(jié)新生RNA的剪接和修飾57二、真核生物mRNA進(jìn)行裝飾性加工真核生物轉(zhuǎn)錄特點(diǎn)：①轉(zhuǎn)錄和翻譯在時(shí)間及空間上是分割的，保證了精細(xì)的調(diào)節(jié)。②合成的RNA鏈經(jīng)過多種加工mRNA5’-cap3’-poly(A)intron去掉exon③核內(nèi)初級(jí)轉(zhuǎn)錄產(chǎn)物大部分需要降解核不均－RNAhnRNA20%被降介。二、真核生物mRNA進(jìn)行裝飾性加工①轉(zhuǎn)錄和翻譯在時(shí)間及空間58三、不同RNA剪接方式導(dǎo)致一個(gè)基因多種產(chǎn)物四、核糖體RNA和tRNA前體的轉(zhuǎn)錄后加工轉(zhuǎn)錄出來(lái)的RNA稱為RNA前體，需要進(jìn)行加工原始轉(zhuǎn)錄產(chǎn)物→成熟RNA分子此過程稱“轉(zhuǎn)錄后加工”。三、不同RNA剪接方式導(dǎo)致一個(gè)基因多種產(chǎn)物59

（四）轉(zhuǎn)錄產(chǎn)物的“加工”（四）轉(zhuǎn)錄產(chǎn)物的“加工”601、核糖體RNA前體的轉(zhuǎn)錄后加工2、tRNA前體的加工3、真核細(xì)胞mRNA前體的加工：真核細(xì)胞mRNA的結(jié)構(gòu)特點(diǎn)P266返回1、核糖體RNA前體的轉(zhuǎn)錄后加工返回61①除去前體5‘和3‘端多余的核苷酸；2、tRNA前體的加工②有些tRNA還需要添加3‘端的－CCA三核苷酸順序；③有些堿需修飾，包括甲基化、脫氨、還原作用等。轉(zhuǎn)錄產(chǎn)物的“加工”返回①除去前體5‘和3‘端多余的核苷酸；2、tRNA前體的加工②62內(nèi)含子（intron）：3、真核細(xì)胞mRNA前體的加工基因的插入順序?yàn)椴荒芫幋a時(shí)DNA順序。外顯子（exon）：基因內(nèi)被內(nèi)因子分隔的編碼片段或編碼的DNA序列。轉(zhuǎn)錄產(chǎn)物的“加工”返回內(nèi)含子（intron）：3、真核細(xì)胞mRNA前體的加工基因的63

mRNA前體的轉(zhuǎn)錄后加工，包括了對(duì)5‘端和3‘端（首、尾部）的修飾以及對(duì)中間的部分進(jìn)行剪接（splicing）。轉(zhuǎn)錄產(chǎn)物的“加工”返回mRNA前體的轉(zhuǎn)錄后加工，包括了對(duì)5‘端和3‘端（首64(1)首、尾的修飾在核內(nèi)經(jīng)首、尾兩部分修飾，在專一的酶促作用下，5‘端形成特殊的帽子結(jié)構(gòu)。P269圖大多數(shù)mRNA的3‘未端有polyA以ATP為底物，在RNA末端腺苷酸轉(zhuǎn)移酶的作用下，在3‘端逐個(gè)添加多聚腺苷酸尾巴。（2）mRNA的剪接P268大多數(shù)真核生物基因都是斷裂基因，斷裂基因的轉(zhuǎn)錄產(chǎn)物需通過拼接轉(zhuǎn)錄產(chǎn)物的“加工”返回(1)首、尾的修飾（2）mRNA的剪接P268轉(zhuǎn)錄產(chǎn)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（帶帽）

2)

3’end=tailing（加尾）3)

Internal=splicing（剪接）4)

Internalmethylation（內(nèi)部甲基化）5)

Editing（編輯）MechanismPost-transcriptionalProcessin67第11章DNA的復(fù)制、修復(fù)和重組-DNATranscr課件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的復(fù)制、修復(fù)和重組-DNATranscr課件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的復(fù)制、修復(fù)和重組-DNATranscr課件79EukaryoticGenesareSplit

Discover:RichardJ.RobertsandPhillipA.Sharp(TheNobelPrizelaureateinPhysiologyorMedicine1993)Introns

intervenebetweenexons

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

EukaryoticGenesareSplitDis80DenatureHybridizewithmaturemRNAAndvisualizeunderEMR-looptechniquesDNAtemplatestrandMaturemRNADenatureHybridizewithmature81第11章DNA的復(fù)制、修復(fù)和重組-DNATranscr課件82第11章DNA的復(fù)制、修復(fù)和重組-DNATranscr課件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的復(fù)制、修復(fù)和重組-DNATranscr課件87第11章DNA的復(fù)制、修復(fù)和重組-DNATranscr課件88Alternativesplicing&Trans-splicing

Alternativesplicing

SplicingispreciseBUT...

Individualpre-mRNAmaybesplicedtomorethanonekindofmRNAbyremovingdifferentcombinationsofintrons/exons

AlternativeformscanberegulatedSexdeterminationinDrosophilaTrans-splicingDefinition-5'and3'junctionsondifferentRNAmoleculesRelativelyrare-otherwiseOccursnaturallyintrypanosomesandinC.elegans-maybeelsewheretoo.Alternativesplicing&Trans-s89AlternativesplicingcangeneratemultipleproteinsfromasinglegeneAlternativesplicingcangener90第11章DNA的復(fù)制、修復(fù)和重組-DNATranscr課件91CappingTailingCappingTailing92Post-transcriptional

processingofpre-rRNAInkaryotes

Cleavage,trimmingandmodificationIneukaryotes

1)

Cleavage,trimmingandmodification2)

Splicing

(SomeeukaryotessuchasTetrahymena-四膜蟲)Post-transcriptional

processi93第11章DNA的復(fù)制、修復(fù)和重組-DNATranscr課件94第11章DNA的復(fù)制、修復(fù)和重組-DNATranscr課件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的復(fù)制、修復(fù)和重組-DNATranscr課件98第11章DNA的復(fù)制、修復(fù)和重組-DNATranscr課件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的復(fù)制、修復(fù)和重組-DNATranscr課件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的復(fù)制、修復(fù)和重組-DNATranscr課件103第11章DNA的復(fù)制、修復(fù)和重組-DNATranscr課件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的復(fù)制、修復(fù)和重組-DNATranscr課件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演講完畢，謝謝觀看！演講完畢，謝謝觀看！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合成與DNA合成的比較：第十三章RNA的代謝

（1）催化方向均是5‘－3‘，延伸的機(jī)理相同：反應(yīng)受焦磷酸水解趨動(dòng)，需要模板。（2）RNA合成不需引物（自身可以獨(dú)立起始合成），且無(wú)外切酶作用（即缺乏核對(duì)能力）；DNA復(fù)制是一個(gè)半保留復(fù)制，RNA合成是全保留的（因是單鏈）。（3）RNA合成起始和終止均受嚴(yán)格的控制，而DNA的終止無(wú)特殊的信號(hào)。RNA合成與DNA合成的比較：第十三章RNA的代謝（1）112CentralDogmaTranscriptionTranslationReplicationReplicationRetro-transcriptionGeneexpressionCentralDogmaTranscriptionT113第11章DNA的復(fù)制、修復(fù)和重組-DNATranscr課件114第一節(jié)依賴DNA的RNA合成轉(zhuǎn)錄的概念

以DNA分子中的某一區(qū)段的一條鏈為模板，在RNA聚合酶的作用下合成一段RNA鏈。5‘――3‘：CodingstrandP3243‘――5：

Templatestrand第一節(jié)依賴DNA的RNA合成以DNA分子中的某一區(qū)115Codingstrand,Sensestrand,CrickstrandTemplatestrand,antisensestrand,WatsonstrandTranscriptionTranslationCodingstrand,Sensestrand,C116原核生物的RNA聚合酶P3241.亞基的生成2.功能：催化RNA的合成:tRNA，rRNA，mRNA3.抑制劑：利福平rifampicin原核生物的RNA聚合酶P3241.亞基的生成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(利鏈霉素)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原核生物的轉(zhuǎn)錄過程

起始階段延長(zhǎng)階段終止階段1、結(jié)合2、解鏈3、引發(fā)4、б因子解離5、核心酶移動(dòng)6、形成3‘，5‘－磷酸二酯鍵7、因子識(shí)別終止信號(hào)8、核心酶停止轉(zhuǎn)錄9、RNA鏈釋放出來(lái)原核生物的轉(zhuǎn)錄過程 起始階段延長(zhǎng)階段終止階段1、結(jié)合2、解鏈124

RNA聚合酶（2ββ‘б）與啟動(dòng)子（promoter）結(jié)合，б組別啟動(dòng)子部位（-35啟動(dòng)子部位）。б和β‘起連接作用。結(jié)合

RNA聚合酶合成RNA的方向?yàn)?‘－3‘，所以從轉(zhuǎn)錄起始點(diǎn)沿RNA聚合酶運(yùn)動(dòng)的方向稱為下游（downstream），核苷酸殘基編號(hào)依次為+2，+3…，而反方向?yàn)樯嫌危╱pstream），核苷酸殘基編號(hào)為-1，-2…。原核生物的轉(zhuǎn)錄過程RNA聚合酶（2ββ‘б）與啟動(dòng)子（promo125

解開一小段DNA雙螺旋，以便產(chǎn)生單鏈DNA轉(zhuǎn)錄模板。解鏈原核生物的轉(zhuǎn)錄過程解開一小段DNA雙螺旋，以便產(chǎn)生單鏈DNA轉(zhuǎn)126

第一個(gè)核苷三磷酸上去（GTP、ATP）（模板）若第一個(gè)是C，那么是PPPG（GTP）結(jié)合上去。

引發(fā)б因子的作用：識(shí)別啟動(dòng)子部位識(shí)別啟動(dòng)部位原核生物的轉(zhuǎn)錄過程第一個(gè)核苷三磷酸上去（GTP、A127DetailedTranscriptionalMechanismThree-stepprocess

1)Initiation

2)Elongation

3)TerminationDNAtranscriptioninprokaryotesDNAtranscriptionineukaryotesInvitroDNAtranscriptionDetailedTranscriptionalMecha128ProkaryoticDNATranscriptionInitiation

1)whatispromoter?

2)howtodetermineth