分子生物学Chapter 8 Eukaryotic RNA TranscriptionWord文档下载推荐.docx
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Eukaryoticpolymeraseshas12ormoredifferentsubunit.
–AllcontainsubunitshomologoustothesubunitsoftheE.coliRNAPolcore(2’).
–atleastfiveothersubunitsarecommontothreedifferentRNAPols.
–EachRNAPolcontainadditionalfourorsevenspecificsubunit.
Allthreepolymeraseclasses....
•weigh>
500,000D
•contain12-16subunits
-someconservedacrossevolution
b’-like(~200,000D)b-like(~140,000D)a-like(~40,000D)
-somesharedamongall3polymerases-someunique
Figure10-26.SchematicrepresentationofthesubunitstructureofyeastnuclearRNApolymerasesandcomparisonwithE.coliRNAcorepolymerase.Allthreeyeastpolymeraseshavefourcoresubunitsthatexhibitsomehomologywiththeβ,β′,andαsubunitsinE.coliRNApolymerase.Thelargestsubunit(L′)ofRNApolymeraseIIalsocontainsanessentialC-terminaldomain(CTD).RNApolymerasesIandIIIcontainthesametwononidenticalα-likesubunits,whereaspolymeraseIIhastwocopiesofadifferentα-likesubunit.Allthreepolymerasessharefiveothercommonsubunits(twocopiesofthelargestofthese).Inaddition,eachyeastpolymerasecontainsfourtosevenuniquesmallersubunits.
1.2TranscriptionFactors
TheinitialfindingthatpurifiedeucaryoticRNApolymeraseenzymescouldnotinitiatetranscriptioninvitroledtothediscoveryandpurificationofadditionalproteins,thegeneraltranscriptionfactors,requiredforthisprocess.Theseproteinswerenotsimplymissingsubunitsofthepolymerase;
theyhadtoassembleintoacomplexontheDNAatthepromoterinordertorecruittheRNApolymerasetothissite
transcriptionfactors:
proteinsrequiredforbringingpolymerasetotranscriptionstartsitetoformapreinitiationcomplex,maynotmigratewiththepolymerasedowntheDNAtemplate,arenotassociatedwithinactivepolymerase
whichareoftwotypes:
A)Upstreamfactors:
bindupstreamsitestoincreaseefficiencyofinitiation...
regulationofWHICHpromotersareused,butnoTemporalorTissuespecificity
TheRNApolymeraseresultingfromtheCoreEnzymeplustheTFUpstreamTranscriptionFactorsisusuallycalledtheBasalApparatus...orHoloEnzyme,forcomparisontoprokaryoticRNApolymerases...
B)Induciblefactors:
bindupstreamanddownstreamsites;
Time&
Tissuespecificity
TheseInducibleFactorshavenocognatesinprokaryoticRNApolymerases
1.3DNAGeneExpression(Transcription)Sites
Promoter
Upstreamregulatoryelement
1.4Theprocessoftranscription
•Transcriptionbyeachpolymeraseinvolves:
1)recognitionofpromotersequencesandassemblyofaninitiationcomplexatthetranscriptionstartsite
2)promotermelting
3)initiationandpromoterescape
4)elongation(RNAsynthesis)
5)termination
2.PolItranscription
(Reading:
Weaverchapter10pp.286-288;
chapter11pp.325-331;
chapter16pp.498-501
Overview:
PolIisresponsiblefortranscriptionofmajorribosomalRNAs(note:
5SRNAistranscribedbypolIII):
•18SRNA•28SRNA•5.8SRNA
vaccountsfor50-70%oftotalRNAsynthesis-
v•producesasingletranscriptthatisprocessedtogenerateallthreeRNAs
v•rRNAgeneismulticopy(hundreds-thousandsofcopies)
2.1Genestructure
hundreds-thousandsofcopiesoftheidenticaltranscriptionunit,containing18S-5.8S-28SRNA.
RibosomalRNAsarewellconservedineukaryotes,spacers(includinglength)arenot.
NucleolarendonucleasesprogressivelycleavetheprimarytranscripttoyieldthematureRNAs
Productisa45SRNAthatisprocessedtogivethe18S,5.8S,and28SmaturerRNAs
Fig8-5
U3RNAandassociatedproteins(U3snRNP)bindnearthe5’-endoftheprimarytranscriptanddirectprocessing.
2.2PolI
•purifiedfromnucleoli(polIIandpolIIIexcluded)
•unlikepolII(andsometimespolIII),polIisnotinhibitedbya-amanitin
•highelongationrate(30nucleotides/second)
•500-600KD,9-14subunits
2.3Promoterstructure
comparingrRNAgenepromotersfromdifferentspeciesshowsasimilarorganizationbutpoorconservation
considermammaliangenes®
mapelementsbyscanninglinkerinsertions
Experimentsidentifytworegionsthatarerequiredforfulltranscription,correspondtobindingsitesfortwodifferentsetsoffactors
Corepromoter:
-45to+20(+1isStartNuc)...G,C-rich85%seq
UpstreamControlElement(UCE):
-180to-107...G,C-rich
2.4Transcriptionfactors
Twopromoterbindingactivities:
UBF:
upstreambindingfactor
SL-1:
promoterselectivityfactor(Sigma-Likefactor)
AllproteinsthatmakeupUBFandSL-1havebeenrecentlycloned,expressedrecombinantly,showntobesufficientforpolItranscription.
UBF+SL1+polI--minimumfortranscription
WhatareUBFandSL-1?
asingle94-97KDprotein
cloned
bindsUCEandcorepromoter
DNAbindingdomaincharacterized
•resemblesHMG1(chromainprotein)
•bindsinminorgroove
•recognizesstructure,notsequence
•bendslinearDNA,inducespositivesupercoiling
purifiedasamacromolecularcomplex,>
200KD
contains4proteins:
TBP,TAFI110,TAFI63,TAFI48
TAFs(TBPActivationFactors);
species&
promoterspecificity
TBP
TBPispresentinbothTFIIDandSL-1
2.5DNArecognition
InpolIItranscription,TBPbindsDNA(TATA-box)
InpolItranscription,TBPbindsTAFIs,TAFIsbindDNA
•noTATA-boxincorepromoter
•bothTAF48andTAF63canbecross-linkedwithDNA
•TAFI63containstwoCys2-His2zincfingers,mediates
interactionwithcorepromoter
Processoffactorassemblyatthepromoter
2.7Transcriptiontermination
Wellunderstood.IncontrasttopolII:
•terminationoccursatwell-definedsites
•polyadenylationdoesnothappen
Transcriptionterminatesintheintergenicspacer(IGS).
Specificterminationpreventsoneextendingpolymerasefromoccludingdownstreaminitiationcomplexes
Terminationstudiedinmammalian,frog,andyeastsystems.
•aswithinitiation,mechanismisconservedalthoughfactorsarespeciesspecific
•terminationhappensatoneofeighttandemlyrepeatedterminationsites565bpdownstreamofthemature3’-end
•terminationsites,calledSalboxes,sharean18bpconsensussequence:
AGGTCGACCAGATNTCCG
SalIsite
•asingleSalboxissufficientfortermination,servesasthebindingsiteforTTFI(transcriptionterminationfactorI).
•terminationrequiresTTFIbindtheSalboxandthattheSalboxbeinthecorrectorientation
TTFIcausesthepolymerasetoterminate≈10bpupstreamoftheSalbox.Theinvivomatureendis≈21bpupstreamoftheSalbox-apolIassociatedexonucleasecuts≈11bpfromthe3’-end
3.PolIIITranscription
3.1GenestranscribedbyRNApolymeraseIII
SmallRNAsinvolvedinproteinsynthesis
5.8SRNA(ribosomalcomponent)
tRNAs
SmallRNAinvolvedinmRNAprocessing
U6snRNA(spliceosome)
SmallRNArequiredforproteintransport
7SLRNA(signalrecognitionparticle)
RNAinvolvedinDNAreplication
TelomeraseRNA
RNAsinvolvedinviralgeneexpression
VARNAs(Adenovirus)
EBERRNAs(Epstein-Barrvirus)
Unknownfunction
7SKRNA
AluI
B2(Rodent)
SINE
3.2TypesofpolIIIpromoters
Howdoyoumapoutpromoterstructure?
Assayforactivity
-cell-freeextracts-Xenopusoocyteinjection
•Linkerinsertionanalysis•Mutationalanalysis
•Deletionanalysis
5.8SrRNAgene
noupstreamsequencesrequiredfor5SrRNAtranscription!
ThreedifferenttypesofpromotersdirectpolIIItranscription
Type1,Type2genes:
intragenicpromoters,internalcontrolregions(ICRs)arelocateddownstreamoftranscriptionstart
Type3genes:
extragenicpromoters,upstreamcontrolregionsincludingTATAboxes
Note:
thetypedistinctionsarenotabsolute,forinstance
-upstreamsequencesintype1and2genescanaltertranscriptionlevels
-X.laevistRNA(Ser)Sechasbothintra-andextragenicpromoterelements
3.2.1Type1:
e.g.Xenopuslaevis5SRNA
-3typesof5Sgenes:
adult400genes
majoroocyte20,000genes
traceoocyte1,000genes
-ICRspans45bp,runsfrom+50to+90
-3distinctinternalpromoterelements
A-box
C-box
intermediateelement(IE)
3.2.2Type2:
representedbytRNAgenes
-ICR(+10/+65)
-2distinctpromoterelements
A-box-correspondstoD-loopsequence
B-box-correspondstoT-loopsequence
Intype1genes:
A-boxisat+50
Intype2genes:
A-boxisat+10,determinestranscriptionstartsite
SpacingbetweenAandBvaries(30-60nts.)
5SA-boxandtRNAA-boxareinterchangeable
3.2.3Type3genes:
e.g.U6snRNA,7SKRNA
-noICR
-multipleupstreampromoterelements
-TATAbox(-30)
-proximalsequenceelement(-60)
-distalsequenceelement(-200)
DSEismadeupbytwosequencemotifs
-GCbox:
GGGCGG
-octamermotif:
ATGCAAAT[OCTbindsOctamerBindingFactor(OTF-1)
StimulatorysitesforRNAPolIII]
SimilartostructureforotherURNAsthataretranscr