RNA Biosynthesis（transcription）RNA的生物合成课件

RNABiosynthesis(Transcription)RNABiosynthesis（transcription）RNA的生物合成课件11．复制和转录过程有什么相似之处？又各有什么特点？

2.试述真核细胞RNA转录后的加工修饰1．复制和转录过程有什么相似之处？又各有什么特点？

2.2Concept

TheprocessofcopyingDNAtoRNAbyanenzymecalledRNApolymerase(RNAP)ThetransferofgeneticinformationfromDNAintoRNAtranscriptionRNADNA

ConcepttranscriptionRNA3ContentTemplatesandEnzymesTheProcessofTranscriptionPost-transcriptionalModificationContentTemplatesandEnzymes4ThedifferencebetweenreplicationandtranscriptionThedifferencebetweenreplica5SubstancesfortranscriptionSubstrates:

NTP(ATP,UTP,GTP,CTP)

Template:

DNAEnzyme:

RNApolymerase(RNA-pol)OtherproteinfactorsSubstancesfortranscriptionSu6TemplatesandEnzymesSectionISectionI7TemplatefortranscriptionStructuralgene:aDNAfragmentcodingforanyRNAorproteinproductotherthanaregulatoryelementTemplatestrand(sensestrandorWatsonstrand)

:thestrandofDNAthattheRNApolymeraseusesasatemplatetoproducecomplementarymRNACodingstrand(antisensestrandorCrickstrand)

:thestrandthatisnotusedasthetemplateithasthesamebasesequenceastheRNAtranscriptproduced(althoughwiththyminereplacedbyuracil)TemplatefortranscriptionStru85′···GCAGTACATGTC···3′3′···cgtgatgtacag···5′5′···GCAGUACAUGUC···3′N······Ala·Val·His·Val······CCodingstandTemplatestrandmRNAProteintranscriptiontranslation5′···GCAGTACATGTC···3′3′···c95

3

3

5

TemplatestrandCodingstrandCodingstrandTemplatestrandStructuralgeneDirectionoftranscriptionDirectionoftranscription53TemplatestrandCodingstra10Asymmetrictranscription

certainregionofonlyonestrandofDNAservesasatemplatefortranscriptionThetemplatestandisnotalwaysrestrictedtothesamesingleDNAstrandAsymmetrictranscription11RNApolymeraseProkaryoticRNApolymeraseRNApolymeraseProkaryoticRNA12coreenzymeholoenzyme

coreenzymeholoenzyme13ThebindingsiteofRNApolholoenzymeonthetranscriptioninitiationregionThebindingsiteofRNApolhol14EukaryoticRNApolymeraseEukaryoticRNApolymerase15AsiteforRNApoltoidentifyandbindonthetemplateOperon:aunitoftranscriptioninprokaryotesincludingoneormorestructuralgenesandupstreamregulon

5

3

3

5

StructuralgeneregulonRNA-polpromoter:

thesiteonthetemplateDNAforRNApolbindingAsiteforRNApoltoidentify16RNApolymeraseprotectionassay目录RNApolymeraseprotectionassay17TranscriptioninitiationTTGACAAACTGT-35region(Pribnowbox)TATAATPuATATTAPy-10region1-30-5010-10-40-205

3

3

5

Prokaryoticpromoteconservativesequencerecognitionsite

5

5

RNApolprotectionregionStructuralgene3

3

TranscriptioninitiationTTG18

TATAbaxCAATbaxGCbaxenhancerCis-actinelementStructuralgene-GCGC---CAAT---TATATranscriptioninitiationEukaryoticpromoteconservativesequenceTATAbaxCAATbaxGCbaxenhan19TheProcessofTranscriptionSectionIISectionII20InitiationTwoissuesrequiredtobeaddressed

thebindingofRNApolymerasetotheinitialregionofthetranscriptiontemplatepreciselyonlyonestrandofDNA,thetemplatestrand,isusedtoproducemRNAProkaryotictranscriptionInitiationTwoissuesrequired21TheDNAisunwoundandbecomessingle-strandedinthevicinityoftheinitiationsite(definedas+1).RNApolholoenzyme(2)specificallybindingtothepromoterregiononDNA1stpolyreactioncatalyzedbyRNApolandtranscriptioninitiationcomplexisformedRNApol(

2

)-DNA-pppGpN-OH3

transcriptioninitiationcomplex5

-pppG-OH+

NTP

5

-pppGpN

-OH3

+ppiInitiationTheDNAisunwoundandbecome22Elongation

subunitdissociatesfromtheholoenzyme，andelongationproceeds

withtheeffectofthecoreenzyme，NTPmaintainpolymerization，RNAstrandelongated(NMP)n

+

NTP

(NMP)n+1

+PPiElongationsubunitdissociate23transcriptionbubble：RNA-pol（Coreenzyme）

····DA

····RNA目录transcriptionbubble：RNA-pol（24目录目录255

3

DNAFeatherphenomenaduringprokaryotictranscriptionribosomeRNARNApol53DNAFeatherphenomenadurin26Rho(ρ)factordependenttranscriptionterminationRho-independenttranscriptionterminationTerminationTheprocessoftheRNApolymerasestoppingonDNAtemplateandthedissociationoftheRNAfromthetranscriptioncomplexClassificationRho(ρ)factordependenttrans27ATPRho(ρ)factordependenttranscriptionterminationATPRho(ρ)factordependent28TerminationindependentonRhofactorTheterminatorsequencewithintheRNAforthestopofRNApolymerase.Theterminatorsequenceusuallyformsastem-loophairpinstructureleadingtothedissociationoftheRNAPfromtheDNAtemplateTerminationindependentonRho295`UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU...3`5`UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU...3`

RNA5

TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT...3

DNA

UUUU...…

UUUU...…5`UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU...3`stem-loop/hairpinstructure5`UUGCAGCCUGACAAAUCAGGCUGAUGGC30MechanisminvolvedintheterminationbystemloopallostericchangeofRNApolyforthestopoftranscriptiondissociationoftranscriptioncomplexforthereleaseofRNA5´pppG53

35RNA-polMechanisminvolvedintheterm31EukaryotictranscriptioninitiationinitiationAvarietyofupstreamcontrolelementsexistRNA-poldoesnotdirectlybindtothetemplateintheinitiationMuchmorecomplicatedthanprokaryotictranscriptionEukaryotictranscriptioniniti32TranscriptionInitiationnsiteTATAboxCAATboxGCboxenhancercis-actingelementTheupstreamregionfromthetranscriptioninitiationAATAAAExcisionandaddingtailTranscriptionTerminationsiteModificationsiteexonTranslationinitiationsiteintronOCT-1

OCT-1：ATTTGCAToctamerTranscriptionTATAboxCAATboxG33TranscriptionfactorTrans-actingfactors:TheproteinswithabilitytorecognizeandbinddirectlyorindirectlytotheupstreamcontrolelementofDNAHundredsfactorshavebeenreportedTranscriptionalfactors(TF):

Amongthetrans-actingfactors,theproteinswithabilityto

binddirectlyorindirectlytoRNApolTranscriptionfactorTrans-acti34参与RNA-polⅡ转录的TFⅡ

参与RNA-polⅡ转录的TFⅡ35Pre-initiationcomplex(PIC)Eukaryotictranscriptioninitiationrequireavarietyoftranscriptionfactor

becauseof

theindirectlybindingofEukaryoticRNA-poltoDNAPre-initiationcomplex(PIC)36POL-ⅡTFⅡFⅡAⅡBPICtranscriptioncatalyzedbyRNA-PolⅡPOL-ⅡTFⅡFⅡHⅡETBPTAFTFⅡD-ⅡA-ⅡB-DNAcomplexTATAⅡAⅡBTBPTAFTATAⅡHⅡECTD-PAfterPICassemble，CTDphosphorylationbyTFⅡH

POL-ⅡTFⅡFⅡAⅡBPICtranscription37Piecingtheory

Theeukaryotictranscriptionrequires3to5TFbindingtogetherandbecomingthecomplexfortheactivationofspecificgenetranscriptionPiecingtheoryTheeukaryotic38ElongationSimilartoprokaryotictranscriptionelongation.synchronoustranscriptionandtranslationisnotobservedbecauseofsegregationofnuclearmembraneTheforwardmovingprocessofRNA-polencounteringnucleosomeThetranslocationanddissociationofnucleosomesobservedduringthetranscriptionElongationSimilartoprokaryot39RNA-PolRNA-PolRNA-PolnucleosomeNucleosometranslocationduringelongationDirectionoftranscriptionRNA-PolRNA-PolRNA-Polnucleosom405------AAUAAA-5

------AAUAAA--nuclease-GUGUGUGRNA-polAATAAAGTGTGTGModificationsitefortranscriptiontermination5

5

3

33addingtailAAAAAAA······3

mRNATermination——associatedwithpost-transcriptionalmodification5------AAUAAA-5------AAUAAA41Post-transcriptionalmodificationSctionIIISctionIII42ConceptIneukaryoticcells,ageneticprocessfortheconversionofaprimarytranscriptRNAtomatureRNAConcept43MajormodificationssplicingcleavagemodificationadditionMajormodificationssplicingcle44EukaryoticmRNAprocessingModificationstothe“head"(5')and“tail"(3')endsofthemRNA5

capping

(m7GpppGp—)3

polyadenylation

(polyAtail)EukaryoticmRNAprocessingModi455

capstructure5capstructure465pppGp…5GpppGp…pppGppiGMPtransferase5

m7GpppGp…methyltransferase

SAMTheformationof5

cap

5ppGp…phosphatasePi5pppGp…5GpppGp…pppGppiGMP47mRNAsplicinghetero-nuclearRNAa(hnRNA):theprimarymRNAwithinnucleussnRNA:aclassofsmallRNAmoleculeswithinthenucleus

ProteinswithinNuclear

smallnuclearribonucleoproteins

（splicesome）snRNAmRNAsplicinghetero-nuclearR48astrandofDNAcontainingbothintronsandexons.Mostadvancedeukaryoteshaveinterruptedgenesandintronsarelongerthanexons.TheresultinggeneismuchlongerthanitscodingregionInterruptedgene

CABDCodingregionA、B、C、DNon-codingregionastrandofDNAcontainingbot49ExonandintronexontheregionsofagenethatarerepresentedinmatureformofthemessengerRNA(mRNA)intronThesectionsofagenethatarepresentinprecursormRNA(pre-mRNA)andremovedbyaprocesscalledsplicingduringtheprocessingtomatureRNAExonandintronexon50ChickenOvalbuminGene

hnRNAModificationstothe"front"and"back"endshnRNAsplicingMaturemRNAChickenOvalbuminGeneanditsTranscriptionandPost-transcriptionalModification

目录ChickenOvalbuminGenehnRNAMo51Electron-microscopicpictureofthematuremRNAandDNAhybridizationofChickenOvalbuminDNAmRNA目录Electron-microscopicpictureo52Classificationofintrons

Fourclasses:accordingtogenetypesandsplicingmethodsI：mainlyfoundinmitochondria,chloroplast,andcertainlow-leveleukaryoticrRNAgenesII：foundinmitochondria,chloroplast,inmRNAgenesIII：frequentlyfoundinthesplicingafterloopformation，inmostmRNAgenesIV：tRNAgenesandprimarytranscriptionproductionintron，splicingrequiringenzymeandATPClassificationofintronsFour53mRNAsplicingtoremovetheintronsofhnRNAandligatetheexonsSplicesomeisformedbyligationofsnRNPandhnRNA目录①mRNAsplicingtoremovethein54②③UACUACA-AGUGU4U5U6E1E2U1U2UACUACA-AGUGU6E1E2U1、U4、U5②③UACUACA-AGUGU4U5U6E1E2U1U255pG-OH(ppG-OH,pppG-OH)U-OHGpUpGpA1STtransesterificationreaction

2nd

transesterificationreaction

UpAGpUExon1IntronExon2G-OHUpUpGpA剪接过程的二次转酯反应

pG-OHU-OHGpUpGpA1STtransester56DifferentialRNAprocessing:

RNAeditingshowsthatthecodingsequenceofgeneshasavarietyofdifferentialfunctionafterpost-transcriptionprocessingmRNAeditingHumanapoBgene

mRNA（14500Nucleotides）liverapoB100（MW500000）intestinalcellsapoB48（MW240000）mRNAeditingDifferentialRNAprocessing:57Post-transcriptionalprocessoftRNAtRNAprecursorRNApolⅢTGGCNNAGTGCGGTTCGANNCCDNA目录Post-transcriptionalprocesso58RNAaseP、endonuclease目录RNAaseP、endonuclease目录59tRNAnucleotidetransferase

、ligaseATPADP目录tRNAnucleotidetransferase、l60BasemodificationsReducedreactione.g.：UDHUTranslocationwithinnucleotidese.g.：UψDeaminationreaction

e.g.：A

Ie.g.：AAmMethylation（1）（1）（3）（2）（4）目录BasemodificationsReducedreac61transcription45S-rRNAsplice18S-rRNA5.8S