分子生物学-05真核转录

Chapter5.Transcription&RNAProcessinginEukaryoticcells1.RNApolymerase:3typesRNApolymeraseI:innucleolus;rRNAtranscription;themostcellularRNAsynthesis.Didnotinhibitedbya-amanitinRNApolymeraseII:innucleoplasm,heterogeneousnuclearRNA(hnRNA)transcription(theprecursorformRNA).inhibitedbylowconcentrationsofa-amanitin.RNApolymeraseIII:aminorenzymeactivity.innucleoplasm;tRNAsandothersmallRNAs(5SrRNA)transcription.inhibitedornotbya-amanitin.AlleukaryoticRNApolymerasesarelargeproteins,500kD,8-14subunits.RNApolymerasesIIThelargestsubunithasacarboxy-terminaldomain(CTD),whichconsistsofmultiplerepeatsofaconsensussequenceof7aminoacids.ThesequenceisuniquetoRNApolymeraseII.TheRNApolymeraseactivitiesofmitochondriaandchloroplastsaresmaller,andresemblebacterialRNApolymerase.2.PromoterRNApolymeraseIpromoterhastwosequencecomponentsCorepromoter:~70bp.UBF1:asinglepolypeptide,itbindstobothregions(GC-rich),afterwhichSL1canbind.RNApolIthenbindstothecorepromoter.UBF1:SL1:4subunits,onesubunit--TBPinteractswithRNApolymerase.SL1resemblessigmafactor.Deletionanalysisshowsthatthepromoterfor5SRNAgenesisinternal;initiationoccursafixeddistance(~55bp)upstreamofthepromoter.RNApolymeraseIIIhasainternalpromoterRNApolymeraseIIIhasainternalpromoterPromotersforRNApolymeraseIIIconsistofbipartitesequences:boxAseparatedfromeitherboxCorboxB.Ortheymayconsistofseparatedsequencesupstreamofthestartpoint(Oct,PSE,TATA).PromoterstructureofRNApolymeraseIIIThebindingoftheassemblyfactorsTFIIIAandTFIIIC,theinitiationfactorTFIIIB,andRNApolymeraseIII.PromoterstructureofRNApolymeraseIIITFIIIA:azincfinger.TFIIIB:threesubunits.TFIIIC:atleast5subunits,500kD.TFIIIAandTFIIICareassemblyfactors,whoseroleistoassistthebindingofTFIIIBattherightlocation.TFIIIBfunctionsasapositioningfactor,responsibleforlocalizingRNApolymerasecorrectly.LikeSL1atthepolIpromoter,itresemblesasigmafactor.PromoterstructureofRNApolymeraseIIOneupstreampromoter,withrelativelyfixedlocation.TATAbox:~25bpupstream,8bpconsensussequenceconsistsentirelyofAT.TheTATAboxtendstobesurroundedbyGC-richsequences.Itisalmostidenticalwiththe-10sequencefoundinbacterialpromoters.TheminorityofpromotersthatdonotcontainaTATAelementarecalledTATA-lesspromoters.TBP:TATA-bindingprotein,~30kD.TAFs:forTBP-associatedfactors.TheassemblesatthepromoterPromotersforRNApolymeraseIIhaveshortsequenceelementsCAATbox:-75,CAAT.acommonelements,functionsineitherorientation.Toincreasespromoterstrength.GCbox:-90,GGGCGG.Oftenmultiplecopies.ineitherorientation.arelativelycommonpromotercomponent.PromotersforRNApolymeraseIIhaveshortsequenceelementsPromotersforRNApolymeraseIIhaveshortsequenceelements3.Enhancer•bidirectionalelementsthatassistinitiation•functionineitherorientationandinanylocation(upstreamordownstream)relativetothepromoterAnenhancerinthevirusSV40Itcontainstwoidenticalsequencesof72bpeach,repeatedintandem~200bpupstreamofthestartpointofatranscriptionunit.Each72bprepeatcontainsacopyoftheenhancer4.RNAprocessingmRNAprocessing5’Capping3’CleavageandpolyadenylationSplicingPre-mRNAmethylationmRNAeditingSplicingSplicingTheendsofnuclearintronsaredefinedbytheGT-AGrule.(GT-AGrule).Splicingoccursintwostages,inwhichthe5’exonisseparatedandthenisjoinedtothe3’exon.5’Cappingcap0:inalleukaryotes;guanine-7-methyltransferase.cap1:thepredominanttypeofcapinalleukaryotesexceptunicellularorganisms;2’-O-methyl-transferase.cap2:10-15%ofthetotalcappedpopulation.Poly(A)tialadds~200Aresiduestothefree3′-OHendofthemRNA;catalyzedbytheenzymepoly(A)polymerasethepoly(A)-bindingproteins(PABP)bindsonpoly(A)tail,OnePABPmonomerof~70kDisboundevery10-20basesofthepoly(A)tail.Removalofthepoly(A)tailprecedesthedegradationofcertainmRNAs;Theabilityofthepoly(A)toprotectmRNAagainstdegradationrequiresbindingofthePABP.Removalofpoly(A)inhibitstheinitiationoftranslation.However,therearemanyexamplesinearlyembryonicdevelopmentwherepolyadenylationofaparticularmRNAiscorrelatedwithitstranslation.snRNA(smallnuclearRNA)isanyoneofmanysmallRNAspeciesconfinedtothenucleus;severalofthesnRNAsareinvolvedinsplicingorotherRNAprocessingreactions.(100-300basesand105-106moleculespercell.Innaturalstate,theyexistasribonucleoproteinparticles(snRNP).ThespliceosomecontainssnRNASpliceosome:proteinsandRNAsexistedasribonucleoproteinparticles.ThesnRNPsinvolvedinsplicingareU1,U2,U5,U4,andU6.EachsnRNPcontainsasinglesnRNAandseveral(20)proteins.TheU4andU6snRNPsareusuallyfoundasasingle(U4/U6)particle.ThesnRNPstogethercontain~40individualproteins.AlternativesplicinginvolvesdifferentialuseofsplicejunctionsAlternativesplicinginvolvesdifferentialuseofsplicejunctionscis-splicingandtrans-splicingreactionsSplicingusuallyoccursonlyincisbetweenexonscarriedonthesamephysicalRNAmolecule,buttranssplicingcanoccurwhenspecialconstructsaremadethatsupportbasepairingbetweenintrons.rRNAsplicingtRNAprocessingYeasttRNAsplicinginvolvescuttingandrejoiningrRNAprocessing