Chapter-5-transcription-分子生物学双语课程课件

Chapter 5RNA transcriptionCentral DogmaCentral DogmaContentsContents1 1The transcription in prokaryotesThe transcription in prokaryotes2 2The transcription in eukaryotesThe transcription in eukaryotes4 4RibozymeRibozyme RNA processing RNA processing3 3 Reverse transcription Reverse transcription5 5IntroductionIntroductionBasicconceptsTranscription:thesynthesisofRNAonatemplateofDNAbyusingtheRNApolymerase.InitiationElongationTerminationcodingstrand(Sensestrand)&Templatestrand(antisensestrand)AsymmetrytranscriptionBasic concepts Transcriptionunit:asequenceofDNAtranscribedintoasingleRNA,startingatthepromoterandendingattheterminator.TranscriptbubblePromoter:aregionofDNAwhereRNApolymerasebindstoinitiatetranscriptionTerminator:asequenceofDNAthatcausesRNApolymerasetoterminatetranscription.Upstream Startsite(Startpoint)Downstream SubunitCodinggeneNo.MWPositionFunctionrpoA240103CoreenzymemultiplerpoB1155103catalyticrpoC1160103catalyticrpoD13210390103 factorrecognition5.1 The transcription in prokaryotes5.1 The transcription in prokaryotesE.coli5.1.1RNApolymerase4 subunits:2bind to DNAcatalyze RNA synthesisbut has no specificity.5 subunits:2reduces the affinity of RNApolymerase for nonspecific DNAand greatly increases its affinityfor promoterCore enzymeHoloenzymefactorProkaryotes have the same core enzyme,but the factor is different.p Reusable；p Change RNA pol conformationp Recognize promoterp Have no catalytic abilityE.coli RNA Polymerase functionp does not require a primerp does not have the proofreading functionp does not initiate RNA synthesis randomlyp initiates synthesis at promotersTemplate:DNA Substrates:rNTPs Activator:Mg2+.Product:RNA(1960)(1960)(1960)(1960)5.1.2.1ThestartpointPromoter:aregionofDNAwhereRNApolymerasebindstoinitiatetranscription5.1.2Themechanismoftranscription原核生物启动子结构图consensussequencecore promoterTTGACA（Sextama Box）35 site：RNA pol.loosely binding site（Rsite）TATAAT（Pribnow Box）10 site：RNA pol.firmly binding site（B site）Initiation site：+1 siteRNA transcriptional startpoint（I site）A/G35(R)10(B)+1(I)RNA17bp7bpup promoter mutations look more like the consensusdown promoter mutations look less like the consensuslacUV5lacwild-typeTTTACA-18-TATAATTTTACA-18-TATGTTTTGACA-17-TATAATTAGACA-17-TAGATTTAGATA-17-TAGATTTTGACA-17-TATAAT PRMUp-1 PRMwild-typePromoter efficiency can be increased or decreased by mutation 10 Box was 17bp to 35 Box,it is easy to initiate for RNA polClose to 17 bp，up mutation Far from 17 bp，down mutation17bp distance is more important to transcription than the sequence RNA polymerase binds to one face of DNA to find promoterRNA polymerase binds to one face of DNA to find promoter5.1.2.2 The 5.1.2.2 The initiationinitiation of prokaryotic transcriptionof prokaryotic transcription转录起始复合物转录泡上的三元复合物RNApolmovesalongthetemplate，thenRNAelongation，andkeepthetriplex；Intranscriptbubble,DNAhelixopen,andthenclose，RNAelongation;Asthebubbleprogresses,theDNAduplexreformsbehindit,displacingtheRNAintheformofasinglepolynucleotidechain.RNAelongationrateisconstant,anditwillslowdowninGC-richregion.Transcriptiongeneratespositivesupercoilsandnegativesupercoils.5.1.2.3 Elongation8bpLength of DNARNA complex14bpLength of unwind helixBacterial RNA polymerase terminates at discrete sites Formation of a hairpin in the RNA may be necessary.5.1.2.4 Terminationtwo types of terminators in E.coliRhoindependent terminators sequences that terminate transcription by RNA pol in the absence of any additional factors.Rhodependent terminators sequences that terminate transcription by RNA pol in the presence of the rho factor.Rho factorp a 275 kD hexamer pATPdependent helicases prho binds to a rut site on nascent RNA and translocates along the RNA to reach RNA polymerase,where release the RNA from the DNA template at the terminators Rhodependent terminators原核生物依赖因子的终止反应pAntiterminationallowtheRNApolymerasereadthroughaspecificterminatororterminators.pAntiterminationisaregulatoryevent.Antitermination转录终止和抗终止p The antitermination activity of pN is highly specific.p The recognition sites required for pN action are called nut.p When pN recognizes the nut site,it act on RNA polymerase to ensure that the enzyme can no longer respond to the terminator.5.2.1 RNA polymerase5.2.1 RNA polymeraseNameLocationFunctionSensitive to amanitin RNA polnucleoliMost rRNAinsensitiveRNA pol nucleoplasmmRNA&some snRNAVery sensitiveRNA pol nucleoplasmtRNA、5S rRNA、U6 snRNA&small RNAModerately sensitiveRNA pol in eukaryotic nuclei5.2 The transcription in eukaryotes5.2 The transcription in eukaryotespFor premRNA transcription p Located in nucleoplasm p Maximum subunit 240 kD&have specific COOHend named CTD(Carboxyl Terminal Domain)p CTDend:7aa repeats&high frequency phosphorylation TyrSerPProThrPSerPProSerP p High frequency phosphorylation(Ser,Thr of the CTD)during elongation(not initiation）p The CTD is an important target for differential activation of transcription elongation.RNA pol IIpThecompositionofClasspromoter5.2.2 Transcription factors&promoters5.2.2 Transcription factors&promoters 5.2.2.1 For RNA pol basal promoter:TATA box initiator:PyPyAN PyPy upstream element:CATT box,GC box response element：enhancer,silencerInitiation(1)TFIID:A sort of protein complex(TBP&8TAF)TBP(TATA binding protein)Needed for RNApol I,II,III。Very high conserved Cend domain of 180 aaBinds with DNA in minor groove&winds it。Determination initiation starting siteControl UPE effect for basic transcription RNApol II +20TFIIs(General IF)TIC Transcript initiationAssembleTATA +1Winds minor grooveTFII ATFII DpreTIC(2)TFIIA：Binds to TFII D，Enhances TFII D binding to TATA box,and Stabilize the DNATFII D complex.Transcriptional Initiation ComplexTFII F RNA pol IITFII B Basic TIC(3)TFIIB：binds to TFIID and RNA pol II，as a bridging factor allowing recruitment of RNA pol II to TIC together with RAP38 of TFIIF.(4)TFIIF：includes two subunits(RAP38,RAP74),promoting RNA elongation by its helicase.(5)TFIIH:Large complex made up of5 subunits,includes Helicase activity&Kinase activityfor Phosphorylation of CTD of RNA pol II(6)TFIIE:Associate with TFIIH,Kinasecomplete TICTFII ETFII HmRNAPromoter clearanceTranscription startingFE H B D AcompleteTICTATA +1Wilds minor groove TFII ATFII DpreTICTFII F RNA pol IITFII BbasicTICmRNAPromoter clearanceE H B D ATranscription startingcomplete TICEHpClasspromoterUPE core promoterupstream regulator elementscore promoter：31 6，UPE：180 107，enhance transcription efficiency2.2.2 For RNA polUBF（Upstream binding factor）：）：Binding to core element of promoter and UPE p Transcription factorsTBP(TATAbinding protein):ensures that the RNA pol I is properly localized at the start pointTAF(TBPassociated factors):specific for RNA pol I transcription.SL1（Selectivityfactor1）UBF+UPE UBF+Part of core promoter Two UBF interaction causing DNA to form a loopUPE core promoter3 TAF1 TBPSL1UBFUBF prerRNAAllows RNA pol binding complex to initiate RNA pol IPromoter within gene:5S rRNA,tRNA and scRNA Promoter upstream of transcript startpoint:snRNA(TATA,PSE,OCT)2.2.3 For RNA pol pTwotypesofClasspromoterTFIII CA box:TGGCNNAGTGG(Dloop of tRNA)B box:GGTTCGANNCC (TCloop of tRNA)Promoter for pretRNA+1 A box B box tDNAp Conserved regionp Transcription factors TFCbinding to the Abox、BboxTFIII C+1TBP(TATAbinding protein)BRF(TFIIIBRelated Factor)B factor TFIIIBTBPBRFBTFB：Including 3 subunits TFIIIB allows RNA pol III to bind and initiate transcriptiontRNA RNA pol III5.2.3 Enhancerp Enhancer:a eukaryotic control element that can increase expression of a gene.which may be located far from the gene and may be either upstream or downstream.EnhancerPromoterEnhanceexpressionBasicexpressionPositionnotbefixedFixedregionBi-directionalelementMono-directionalelementNotforspecialgeneonlyforspecialgeneThe differences between promoter and enhancerEnhancerSpecifical trans Factor(tranfactor)Specifical trans Factor(tranfactor).+Activation domain of Enhancer complex Activation domain of Enhancer complex.Enhancer ElementEnhancer Element.(100bp)Enhanson(cisE.)Enhanson(cisE.).(5bp)En.Elem.En.Elem En.Elem.250 180coreCTCIITCIsphIIsphIAp3 Ap2 Ap1eg.SV40 Enhancer（179 250）far distance control，no directionmRNA+1GC CAAT TATA PreTICBiscTICpSilencer：DNAsequencesineukaryotestoregulatethepromoterinfardistancetodecreasethetranscriptionrate.pLikeenhancer,butresultsisreverse.2.3.2 Silencer ppreventthepassageofactivatingorinactivatingeffectsarecalledinsulators.pareabletoblockpassageofanyactivatingorinactivatingeffectsfromenhancersorsilencers.pmayprovidebarriersagainstthespreadofheterochromatin.2.3.3 Insulator（1）TATA-TFD complexformation（2）Pre-TICassemble（3）TICcomplex（4）Transcriptioninitiationpknowmuchlessabouttranscriptionterminationineukaryotesthanthatinprokaryotes；prRNAneedsplicingtoformmaturerRNApmRNAalsoneedsplicing，andhalf-lifetimeisshort2.4 Transcription initiation and 2.4 Transcription initiation and termination termination in eukaryotesin eukaryotesVery few RNA molecules are transcribed directly into the final mature RNA product.Most newly transcribed RNA molecules undergo various alternations to yield the mature product.Take place in both prokaryotes and eukaryotes on the major classes of RNA5.3 RNA ProcessingRNA process:Primary RNA undergoes procedure to become mature RNA1)EndonucleasescutatinsideofRNA2 2)Exonucleases onebyone,cutatendsofRNA5GCGUACAAUGCGA3p Removal of nucleotidesThis processing type is common in prokaryotes and eukaryotes1 1)5Cappingm7G2)3Tailingpoly(A)p Addition of nucleotidesModificationsites:baseandriboseMethylationof2-OHinribosomeNucleotidemodificationintRNA p Modification of certain nucleotidesDHU TC I m7G m5C m6A s2C 5.3.1 5.3.1 rRNArRNA ProcessingProcessingrRNAp have GC content of 60%p usually repetitive,depending on the organism,each cell contains from several hundred to over 20,000 copies of rDNA genep rRNA synthesized in nucleolus and was stimulated by low ionic strength&Mg2+p but other RNA synthesized in nucleoplasm and stimulated by high ionic strength&Mn2+E.coli rDNA structurep Seven different operons for rRNA dispersed throughout the genomep Each operon contains one copy of each of the 5S,the 16S and the 23S rRNA sequences.1 to 4 tRNA molecules are also present in the operon.p The initial transcript has a sedimentation coefficient of 30S(approx.6,000nt),and is normally quite shortlived.5.3.1.1 rRNA processing in prokaryotesProcessingstepsofrRNAinE.coli1)TheprimaryRNAfoldsupintoanumberofstem-loopstructuresbybasepairingbetweencomplementarysequences2)TheformationofthissecondarystructureofstemsandloopsallowssomeproteinstobindtoformaRNPcomplextRNA16S23S5StRNA3)Afterthebindingofproteins,modificationssuchas24specificbasemethylationstakeplace4)Thenprimarycleavagetakeplace,mainlycarriedoutbyRNase,toreleaseprecursorsofthe5S,16Sand23Smolecules5)Furthercleavagesat5and3endsofeachoftheseprecursorsbyRNasesM5,M16andM23Prokaryotic Prokaryotic RibosomesRibosomesE.coli ribosomespThesizesoftheseribosomalproteinsvarywidely,from4646aato557aapMostlytheserelativelysmallproteinsarebasicandbindingtoRNATotal36protein25nm(2750kDa)5.3.1.2 5.3.1.2 rRNArRNA Processing in Eukaryotes Processing in EukaryotespTherRNAgenesarepresentinatandemlyrepeatedclustercontaining100ormorecopiesofthetranscriptionunitpExcept5SRNA，theyaretranscriptedinthenucleolusbyRNApolyerasepTheprecursorhasacharacteristicsizeineachorganism,beingabout7,0007,000ntinyeastand13,5003,500nt(47S)inmammalspItcontainsonecopyofthe18Scodingregionandonecopyofthe5.8Sand28SrRNAcodingregions,p The eukaryotic 5S rRNA is transcribed by RNA poly from unlinked genes to give a 121nt transcript which undergoes little or no processing47S1)Theprecursorsfoldsandcomplexswithproteinsafteritisbeingtranscribed,andthistakesplaceinthenucleolus2)Methylationtakesplaceatover100sitestogive2-O-methylribose3)CleavagesETS1&ETS2(theexternaltranscribedspacers)4)CleavagesITS(internaltranscribedspacer)thenreleasesthe20Spre-rRNAandthe32Spre-rRNA5)The5.8Sregionmustbase-pairtothe28 8SrRNAbeforethematuremoleculesareproducedProcessing steps rRNA（47S）For mammalian prerRNA80S ribosome(4500kD)60S(3000kD)40S(1500kD)33proteins18S rRNA49 proteins5S rRNA 5.8SrRNA&23SrRNAEukaryotic Eukaryotic RibosomesRibosomesTypical eukaryote ribosome performs 107 peptide bonds/sProkaryote ribosome only 15 peptide bonds/sTotal49proteins33proteins5.3.2 5.3.2 tRNAtRNA Processing Processing5.3.2.1tRNAProcessinginProkaryotesinE.coliprRNAoperonscontainscodingsequencesfortRNAp In addition,there are other operons that contain up to 7 tRNA genes separated by spacer sequences5.3.2.2 5.3.2.2 tRNAtRNA Processing in Eukaryotes Processing in Eukaryotes1 1)Theprimarytranscriptformsasecondarystructurewithcharacteristicstemsandloops2 2)Theendonucleasesrecognizeandcleaveoffthe5leaderandthetwo3nucleotides3)TheenzymetRNAnucleotidyltransferaseaddsthesequence5-CCA-3tothe3endtogeneratethemature3endofthetRNAExample:Yeast tRNATyr4)The intron is removed by endonucleolytic cleavage at each end of the intron followed by ligation of the half molecules of tRNApIn prokaryotes tRNA The CCA sequence in the mature 3end is encoded by geneThe difference of tRNA processing between eukaryotes and prokaryotespIn eukaryotes tRNA 3end CCA sequence is added by tRNA nuleotidyltransferase 5.3.3 mRNA Processing 5.3.3 mRNA Processing 5.3.3.1InprokaryotespmRNA do not need process，mRNA transcriptioncouplingwithtranslationpmRNA is transcribed,translated,and degradedsimultaneouslyinbacteria5.3.3.2 In eukaryotes5.3.3.2 In eukaryotespPre-mRNAsfromRNApolymeraseIIallcalledheterogenousnuclearRNA(hnRNA）phnRNAbindtospecificproteintoformhnRNPppre-mRNAsarenotexporteduntilprocessingiscomplete,hnRNPsarefoundonlyinthenucleus.nMostsnRNAtranscribedbyRNApolIInRapidlycoveredwithspecificproteinstoformsnRNPnThemajornucleoplasmicsnRNPscontainindividualsnRNAsandcommonsetsof8proteinswhicharesmallandbasicsnRNA（small nuclear RNA）Overview of eukaryotic mRNA processingOverview of eukaryotic mRNA processing5 pppXpY(30 Nt)3(preRNA)pippXpYGTPSAMpp 5XpYp G 5 ppp 5 XpYpSAH ppiG 5pm7(with Cap0)pp 5 5 CappingCapping(with Cap1)SAMSAMm7G 5 ppp 5 XmpYpm7G 5 ppp 5 XmpYmp G 5 ppp 5 XpYpm7SAHSAH(with Cap2)SAM;S Adenosyl L methionine S-腺苷腺苷-L-甲硫氨酸甲硫氨酸SAH;S Adenosyl L homocysteine S-腺苷腺苷-L-高半胱氨酸高半胱氨酸 Protect premRNA from 5exonuclease Splicing and transfer mRNA to cytoplasm Improve translation efficiencyFunction of cap structurepp 3 Tailing 3 Tailing Cleavage and Cleavage and polyadenylationpolyadenylationPoly(A)tailat3endthecommonfeatureofmaturemRNAThepolyadenylationsignal:5-AAUAAA-3Followedbya5-5-YA-3inthenext11-20nt(Y-pyrimidine)DownstreamaGU-richsequenceGene VIIIRichard John RobertsPhillip Allen Sharp1993 Noble Price WinnersIn Physiology or MedicinepRichard Roberts and Phil Sharp shared the 1993 Nobel Prize for the discovery of the split gene theory.pExperiment material:adenoviruspRichard Roberts Identify the DNA promoter region by sequencing the 5 end of the mRNA and then mapping it to the DNA.The promoter would be upstream of the 5 end of the mRNA.pPhil Sharp Using RNA/DNA hybrids,speculated that the long nuclear RNAs were processed into shorter mRNAsp Split Gene Conception offerednIntronnon-codingsequencenExoncodingsequencenRNAsplicingpp Splicing SplicingSplicingalsorequiresasetofspecificsequencestobepresentexonorintron?Where to cut?Which?IntronsBranchpoint sequence5GU35AG3two transesterification reactionsstep 1step 2phosphodiester bondphosphodiester bondSplicesomeSplicesomepThespliceosomecontainssnRNAspsnRNAanyoneofmanysmallRNAspeciesconfinedtothenucleus;severalofthesnRNAsareinvolvedinsplicingorotherRNAprocessingreactions.psnRNP=snRNA+proteinpSplicingutilizesaseriesofbasepairingreactionsbetweensnRNAsandsplicesites.Splicing?Splicing?SplicingisusuallyoccurredineukaryotesWhy?EvolutionIntronearly?Intronlate?Why?DiscussionnSplicingonlydependsonrecognitionofpairsofsplicejunctions.nAll5splicesitesarefunctionallyequivalent,andall3splicesitesarefunctionallyequivalent.Splice junctions are read in pairs tRNA intron seeds splicingpp Alternative splicing Alternative splicingDefinition：Onepre-mRNAproducesmorethanonemature mRNApp trans splicing trans splicingn Splicing reactions usually occur only in cis between splice junctions on the same molecule of RNA.n trans-splicing occurs in trypanosomes and worms图5-28顺式剪接和反式剪接naprocessinwhichDNAinformationchanges at the level of mRNA.nTypes1substitutionofanindividualbase2basesaddedordeletednFirstlydiscoveredfromthetrypanosomeinmiddleageof1980sAn unusual form of RNA processingpp RNA editing RNA editing4 4 RibozymeRibozyme&catalytic RNA&catalytic RNApWhat is the enzyme?And what is the ribozyme?catalyticRNA,cancatalyzeparticularbiochemicalreactionswithRNAmoleculealone pFirstlydiscoveredin197070s,whenresearchonhowtheintronofrRNA removalinTetrahymena thermophilaself-splicing I intron selfsplicingII intron selfsplicingRNaseRNase P PpRNaesPisanendonucleasecomposedofoneRNAmoleculeandoneproteinmoloeculeVery simple RNP！pThealoneRNAcomponentofRNasePcanworkasanendonucleasein vitropin vitroRNasePreactionrequiresahigherMg2+concentrationthanoccursin vivopNew conception!catalyticRNAworkwithoutproteincomponentproteinmaybeplayanhelperpRetroviruspReversetranscriptaseisanenzymethatusesatemplateofsingle-strandedRNAtogenerateadouble-strandedDNAcopy.5 Reverse transcript5 Reverse transcriptHIV