分子生物学Chapter 8 Eukaryotic RNA Transcription.docx

分子生物学Chapter 8 Eukaryotic RNA Transcription.docx

《分子生物学Chapter 8 Eukaryotic RNA Transcription.docx》由会员分享，可在线阅读，更多相关《分子生物学Chapter 8 Eukaryotic RNA Transcription.docx（26页珍藏版）》请在冰豆网上搜索。

分子生物学Chapter8EukaryoticRNATranscription

Chapter8EukaryoticRNATranscription

Introduction

Figure8-1DiagramofEukaryoticGene

1.BasicFeaturesofEukaryotictranscriton

1.1Threedifferentclassesoftranscription

ThreedifferentRNApolymerasesareresponsibleforgenetranscription:

RNApolymeraseI（polI）:

•ribosomalRNAs（5.8S,18S,28SrRNA）

RNApolymeraseII（polII）:

•mRNAs•somesmallnuclearRNAs（snRNAs）

RNApolymeraseIII（polIII）:

•tRNAs•5SRNA•somesnRNAs•smallcytoplasmicRNAs（scRNAs）

Basicpropertiesofeachpolymerase:

Enzymelocationrelativeactivitya-amanitinsensitivity

PolInucleolus50-70%notinhibited

PolIInucleoplasm20-40%inhibited

PolIIInucleoplasm≈10%species-specific

RNApolymerasesubunits

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?

UBF:

asingle94-97KDprotein

cloned

bindsUCEandcorepromoter

DNAbindingdomaincharacterized

•resemblesHMG1（chromainprotein）

•bindsinminorgroove

•recognizesstructure,notsequence

•bendslinearDNA,inducespositivesupercoiling

SL-1:

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