分子生物学05真核转录

Chapter5.Transcription&RNAProcessing

inEukaryoticcells​Chapter5.Transcription&R11.

RNApolymerase:3types

RNApolymeraseI:innucleolus;rRNAtranscription;themostcellularRNAsynthesis.Didnotinhibitedbya-amanitin

RNApolymeraseII:innucleoplasm,heterogeneousnuclearRNA(hnRNA)transcription(theprecursorformRNA).inhibitedbylowconcentrationsofa-amanitin.

RNApolymeraseIII:aminorenzymeactivity.innucleoplasm;tRNAsandothersmallRNAs(5SrRNA)transcription.inhibitedornotbya-amanitin.

AlleukaryoticRNApolymerasesarelargeproteins,>500kD,8-14subunits.

​1.RNApolymerase:3typesRN2RNApolymerasesIIThelargestsubunithasacarboxy-terminaldomain(CTD),whichconsistsofmultiplerepeatsofaconsensussequenceof7aminoacids.ThesequenceisuniquetoRNApolymeraseII.

TheRNApolymeraseactivitiesofmitochondriaandchloroplastsaresmaller,andresemblebacterialRNApolymerase.​RNApolymerasesIIThelargest32.Promoter

​2.Promoter​4

RNApolymeraseIpromoterhastwosequencecomponentsCorepromoter:~70bp.UBF1:asinglepolypeptide,itbindstobothregions(GC-rich),afterwhichSL1canbind.RNApolIthenbindstothecorepromoter.UBF1:

SL1:4subunits,onesubunit--TBPinteractswithRNApolymerase.SL1resemblessigmafactor.​RNApolymeraseIpromote5Deletionanalysisshowsthatthepromoterfor5SRNAgenesisinternal;initiationoccursafixeddistance(~55bp)upstreamofthepromoter.

RNApolymeraseIIIhasainternalpromoter

​Deletionanalysisshowsthatt6

RNApolymeraseIIIhasainternalpromoter

PromotersforRNApolymeraseIIIconsistofbipartitesequences:boxAseparatedfromeitherboxCorboxB.Ortheymayconsistofseparatedsequencesupstreamofthestartpoint(Oct,PSE,TATA).​RNApolymeraseIIIhasainte7PromoterstructureofRNApolymeraseIIIThebindingoftheassemblyfactorsTFIIIAandTFIIIC,theinitiationfactorTFIIIB,andRNApolymeraseIII.​PromoterstructureofRNApoly8PromoterstructureofRNApolymeraseIIITFIIIA:azincfinger.TFIIIB:threesubunits.TFIIIC:atleast5subunits,>500kD.TFIIIAandTFIIICareassemblyfactors,whoseroleistoassistthebindingofTFIIIBattherightlocation.TFIIIBfunctionsasapositioningfactor,responsibleforlocalizingRNApolymerasecorrectly.LikeSL1atthepolIpromoter,itresemblesasigmafactor.​PromoterstructureofRNApoly9

PromoterstructureofRNApolymeraseII

Oneupstreampromoter,withrelativelyfixedlocation.TATAbox:~25bpupstream,8bpconsensussequenceconsistsentirelyofAT.TheTATAboxtendstobesurroundedbyGC-richsequences.Itisalmostidenticalwiththe-10sequencefoundinbacterialpromoters.

TheminorityofpromotersthatdonotcontainaTATAelementarecalled

TATA-lesspromoters.

TBP:TATA-bindingprotein,~30kD.TAFs:forTBP-associatedfactors.

​PromoterstructureofRNApol10Theassemblesatthepromoter

​Theassemblesatthepromoter11PromotersforRNApolymeraseIIhaveshortsequenceelements

CAATbox:-75,CAAT.acommonelements,functionsineitherorientation.Toincreasespromoterstrength.

GCbox:-90,GGGCGG.Oftenmultiplecopies.ineitherorientation.arelativelycommonpromotercomponent.

​PromotersforRNApolymeraseI12​​13PromotersforRNApolymeraseIIhaveshortsequenceelements

​PromotersforRNApolymeraseI14PromotersforRNApolymeraseIIhaveshortsequenceelements

​PromotersforRNApolymeraseI153.Enhancerbidirectionalelementsthatassistinitiationfunctionineitherorientationandinanylocation(upstreamordownstream)relativetothepromoter

AnenhancerinthevirusSV40

Itcontainstwoidenticalsequencesof72bpeach,repeatedintandem~200bpupstreamofthestartpointofatranscriptionunit.Each72bprepeatcontainsacopyoftheenhancer

​3.Enhancerbidirectionaleleme164.RNAprocessing​4.RNAprocessing​17

mRNAprocessing5’Capping3’CleavageandpolyadenylationSplicingPre-mRNAmethylationmRNAediting​mRNAprocessing5’Capping18Splicing

​Splicing​19Splicing

TheendsofnuclearintronsaredefinedbytheGT-AGrule.(GT-AGrule).​SplicingTheendsofnu20​​21Splicingoccursintwostages,inwhichthe5’exonisseparatedandthenisjoinedtothe3’exon.

​Splicingoccursintwo22​​235’Cappingcap0:inalleukaryotes;guanine-7-methyltransferase.

cap1:thepredominanttypeofcapinalleukaryotesexceptunicellularorganisms;2’-O-methyl-transferase.cap2:<10-15%ofthetotalcappedpopulation.

​5’Cappingcap0:inall24Poly(A)tialadds~200Aresiduestothefree3′-OHendofthemRNA;catalyzedbytheenzymepoly(A)polymerasethepoly(A)-bindingproteins(PABP)bindsonpoly(A)tail,OnePABPmonomerof~70kDisboundevery10-20basesofthepoly(A)tail.Removalofthepoly(A)tailprecedesthedegradationofcertainmRNAs;Theabilityofthepoly(A)toprotectmRNAagainstdegradationrequiresbindingofthePABP.Removalofpoly(A)inhibitstheinitiationoftranslation.However,therearemanyexamplesinearlyembryonicdevelopmentwherepolyadenylationofaparticularmRNAiscorrelatedwithitstranslation.​Poly(A)tial​25snRNA

(smallnuclearRNA)isanyoneofmanysmallRNAspeciesconfinedtothenucleus;severalofthesnRNAsareinvolvedinsplicingorotherRNAprocessingreactions.(100-300basesand105-106moleculespercell.Innaturalstate,theyexistasribonucleoproteinparticles(snRNP).ThespliceosomecontainssnRNA

Spliceosome:proteinsandRNAsexistedasribonucleoproteinparticles.ThesnRNPsinvolvedinsplicingareU1,U2,U5,U4,andU6.EachsnRNPcontainsasinglesnRNAandseveral(<20)proteins.TheU4andU6snRNPsareusuallyfoundasasingle(U4/U6)particle.ThesnRNPstogethercontain~40individualproteins.

​snRNA(smallnuclearRNA)isa26​​27Alternativesplicinginvolvesdifferentialuseofsplicejunctions

​Alternativesplicinginvolves28Alternativesplicinginvolvesdifferentialuseofsplicejunctions

​Alternativesplicinginvolves29cis-splicingandtrans-splicingreactionsSplicingusuallyoccursonlyincisbetweenexonscarriedonthesamephysicalRNAmolecule,buttranssplicingcanoccurwhenspecialconstructsaremadethatsupportbasepairingbetweenintrons.​cis-splicingandtrans-splicin30rRNAsplicing​rRNAsplicing​31​​32tRNAprocessingYeasttRNAsplicinginvolvescuttingandrejoining​tRNAprocessingYeasttRNAspli33rRNAprocessing​rRNAprocessing​34生命没有回头路，