分子生物学 第4章学习资料

第4章RNA转录

(RNAtranscription)

4.1.

基本概念4.2..原核生物RNA转录启动子

4.3.真核生物RNA转录启动子4.4.TranscriptionElongation4.5.Transcriptiontermination4.6.Anti-terminationinRho-dependentterminatorofProkaryotes

4.7.Pre-RNAprocessinginEukaryotes4.1.

基本概念

●

基因表达的第一步●以D.S.DNA中的一条单链作为转录的模板●

在依赖DNA的RNA聚合酶的作用下

●

模板单链DNA的极性方向为3’→5’,而非模板单链

DNA的极性方向与RNA链相同，均为5’→3’.DNA(书写DNA序列时，仅写非模板序列，可不注明极性方向)3’TACTCAT5’RNA5’AUGAGUA3’5’ATGAGTA3’Non-template(codingstrand编码链,sensestrand有义链)template(模板链antisensestrand反义链)若干基本概念

●

按AU，CG

配对的原则，合成RNA分子●一个基因只以一条单链DNA为模板进行转录（不对称转录）

●

RNA的转录包括promotion,elongation,termination

三过程

●从启动子（promoter）到终止子（terminator）称为转录单位

（transcriptionalunit）

●原核生物中的转录单位多为polycistroninoperon

●转录原点记为+1，其上游记为负值，下游记为正值

真核生物中的转录单位多为monocistron,Nooperon

+1-10

+10upstreamstartpointdownstream原核生物往往把负责某一代谢途径、功能相关的一组基因前后相连成串排列、由一个共同的控制区进行转录调控。由这些结构基因和调控区以及调节基因的整个核苷酸序列叫做操纵元(operon).

4.2.RNApromotioninprokaryots

4.2.1.Promoter的结构与功能

(Prok.E.coli

)

a)promoter由两个重要部分组成

●-170~-60Fissite;

bindingsitesforthetranscription-activatorproteinFis.they,donotbindtoRNApolymerase,arenotclassicalpromoterelements,butinsteadaremembersofenhancers●-60-70~-40upstreamcontrolelement(上游控制因子)CAP—cAMPbindingsite(ActivatorSite)●-35~-10corepromoter

RNApol.bindingsite

基因表达调控的正控制位点CAP;CatabolitegeneActivatorProteincAMPAcceptorProtein(环化AMP受体蛋白)(降解物基因激活蛋白)

分解代谢操纵子的激活蛋白PromoterregionidentifiedbyDNAaseI

footprintingmethod

RNApolymerase(noNTP)DNAsequencing

DNaseI

digestionDNA+dissolve

Corepromoterregion(核心启动子)

including

SextamaBox;

RNApol.recognitionsite(Rsite)TTGAC(SextamaBox)-35siteRNApol.looselybindingsitePribnowBox;

TATAAT(pribnowBox)

-10siteRNApol.firmlybindingsite(Bsite)Initiationsite;

+1RNAtranscriptionalstartpoint(Isite)

A/G-35(R)-10(B)+1(I)RNAR-35

B

-10I+1SextamaBoxPribnowBoxInitiationUpelementCAP-cAMPbindingsite(ActivatorSiteAs)

●

AsI+CAP-cAMP●AsI;

IR是CAP-cAMP

的强结合位点（-70~-50）●

AsII;

CAP-cAMP

的弱结合位点（-50~-40）cooperativeeffect提高AsII

的结合效率IR-70AsI-40AsII

IR

-50

RNApol

AsII+CAP-cAMP

复合体促使SextamaBox附近GC岛区的双螺旋结构稳定性降低，

PribnowBox的解链温度降低，利于转录启动

AsI

AsIIGCIslandSextama

Pribnow

●NullAsII→

RNApol.intopribnowBoxButtranscriptionoff

●

AsII+CAP-cAMP

promotionRNApol.intoSextamaBoxintoPribnowBoxstartingtranscription

RNApol

RNApolc)PribnowBox的突变与遗传效应

T82T84G78A65C54A45T80A95T45A80A50T96c(A/G)t(100)RBI

λ-C17

TATAATGλ-pREAAGTATCAC

LRNA-strTAAAATCBio-p98TAAATT

C

LRNA-tyr

TATGAT

CCgalTATGGT

T

14/16downmutationλ-ClacTATGTTAALRNA-trp

TTAACTCLac-p115TATTGTAλ-CiR

TACACT

C

GC2/16upmutation●PribnowBox中A/T→T/A,改变了碱基堆积状况(嘌呤到嘧啶大于嘧啶到嘌呤),

改变了RNApol.与模板链的结合效率转录效率.

●ProbnowBoxA/T→G/C

双螺旋体稳定性加强,转录率下降l

SextamaBox与PribnowBox间距17bp,

有利于RNApol启动间距趋近于17bp，upmutation间距远离于17bp，downmutation17bp的间距较17bp的序列对转录更为重要l

SextamaBoxorPribnowBoxmut.SextamaBoxandPribnowBoxmut.→

转录率下降100X→

转录率下降1000X4.2.2.RNApolymerase

inprokaryotes原核生物的RNA聚合酶RichardBurgessandAndrewTravers(1969)150kD160kD

70kD

40kDseparationofthesubunitsofE.coliRNApolymerasebySDS4.2.2.RNApolymerase

inprok.σβ

αα

β’CoreEnzymeforelongationforinitiation依靠静电作用力依靠空间结构非专一性与非特异DNA结合专一性与特异DNA结合cover60bp

HoloEnzyme

σαβα

β’全酶与DNA的非特异性结合力下降了一万倍,结合常数为107/mol,半衰期在一秒钟以下,但是全酶依靠空间结构的特异性对启动子的结合能力大大提高,结合常数为1014/mol,半衰期:数小时.依靠蛋白质中碱性基团与DNA的磷酸根之间的静电作用力结合,是一种与DNA序列无关的非特异性结合.结合常数；1011/mol,半衰期:60’σ因子；★重复使用(Re-usable)

★使Holo-enzyme识别SextamaBox,与模板链结合

★修饰RNApol

构型降低全酶与DNA的非专一性结合力

增强全酶与R,Bsite的专一性结合力

导致RNA链的延伸缓慢Hybridization-competition(Ekkehard

Bautz)RNAistranscribedfromregionXIfonlyYRNAcompetitorisadded,nocompetitionisseen被标记的转录物与相应的RNA-competitor才能表现为“competition”

被标记的转录物特异性越高，表现的“competition”效应越明显Holoenzyme

(withσsubunit)ishighlyspecificforimmediateearlyanddelayedearlygenesoftranscriptionT4competitorRNAsisolatedfromE.coilcells1min(green),5min(red),and17min(blue)afterinfectionwithT4phageBycontrastcoreenzyme(withoutσsubunit)hasnospecificity

immediate

earlyDelayedearlylateimmediate

EarlyDelayedearly检测双链转录CoreenzymewithoutσsubunitactsinanunnaturalwaytranscribingbothDNAstrands两条互补配对的RNA分子，对RNase有抗性如果在体外T4DNA以非对称方式正确地转录形成T4RNA，那么在体内转录的T4RNA与体外转录的T4RNA不能互补，也就不能形成双链RNA分子。体外如果T4RNA是以对称方式合成的，则会有一半的体外转录产物与体内RNA互补，形成对RNase具有抗性的杂交分子。Bautz以T4噬菌体DNA在体外由RNA聚合酶全酶/核心酶转录

SigmastimulatestightbindingbetweenRNApolymeraseandpromoterH3-T7DNA+HoloE/CoreEExcessunlabeledT7DNAHoloE/CoreEdissociatedfromDNA-E,thenrebindFilteredmixturesthroughNitrocelluloseMonitorH3-T7DNA/Eretained

复合体紧密与半衰期程度相关highertemperaturefavorstighterbindingbetweenRNApolymeraseholoenzymeandT7DNA(37co)

?Holoenzymeand3H-labeledT7DNA)γ-P32ATPorγ-P32GTP?!σfactorstimulateinitiationoftranscriptionCoreE+14C-ATPmeasuredbulkRNAsynthesis,orelongationσcouldappeartostimulateelongationbystimulationinitiationandprovidingmorechainsforcoreEtoelongation!!14C-ATP?

OverallshapesofE.coliRNApolymerasecore(a)andholoenzyme(b)deducedfromelectronmicroscopyCoreEHoloELooslybindtighterbindingααα因子；★促使RNApol与

CAP（activationregion）-cAMP（inAsI

、AsII）结合

★位于前端的α因子使双链解链为单链

★位于尾端的α因子使单链重新聚合为双链

★核心酶的组建因子

α+α→2α+β

→+β’

α因子CTD与CAPARI结合

α因子NTD与CAPARII结合

激活转录，准确起始RichardGourse(Science262(26Nov1993)

w.t.

promoter

verystrongrrnBP1promoterregion

(-88to+1),

mut.

Promoter(AsI,AsII)

(SUB:anirrelevantseq.insteadofUPfrom-59to-41)(-41:lackedtheUPupstreamof-41andvectorseqinstead)

transcribedwiththreedifferentRNApolymerases(RNA-P),

(1)wild-typepolymerasewithanormalα-subunit;

(2)α-235,α-subunitwasmissing94aafromC-terminus;(3)R265C,α-subunitcontainedatposition265.

Cysteine(C)inplaceofthenormalArginine(R)

includedalabelednucleotidetolabeltheRNAgelelectrophoresisperformedautoradiographytovisualizetheRNAproducts

ImportanceoftheαinUPelementrecognitionW.TRNApolα-mutofRNApolW.T+mut

α-mut

promoterw.tUPdel.UPdel.w.tUPdel.

Importanceoftheαsubunit

inUPelementrecognitionβ因子；★促进RNApol+NTP→RNAelongation

★完成NMP之间的磷酸脂键的连接

★Editing

★与

Rho(ρ)因子竞争RNA3’-end

★构成HoloEnzyme后，β因子含有

两个位点Isite(RifS);Esite(RifR);要求高浓度的ATPorGTP专一性地结合ATPorGTP对

NTP非专一性结合β’

因子；★强碱性亚基★促使RNApolymerase与非模板链（sensestrand）结合

★受K酶抑制

HoloEnzyme含有五个功能位点

★sensestrandDNAbindingpoint(β’)

★DNA/RNAhybridsite(β)

★D.S.DNAunwindingpoint(α)

★D.S.DNArewindingpoint(α)

★

σfactorpoint

原核生物RNApol(Core)的结构与功能EnzymeMovementDNAcodingstrand(β’

)Rewindingpoint(α)Unwindingpoint(α)RNAbindingsiteRNA/DNAhybrid(β)DNAtemplatestrandCoreEnzyme使

DNA形成10-17bp的解链区

原核生物RNApol(Core)的结构与功能EnzymeMovementRNAbindingsiteRNA/DNAhybrid(β)IEIsite(RifS);Esite(RifR);非专一性结合

NTP专一性地结合ATPorGTPSeparationandreconstitutionofRNApolymerasePurificationoftheindividualsubunitsofE.coliRNApolymeraseinUreaelectrophoresisbuffer

(Alfredheil&Ziling1970)locatethedeterminationofRifampinresistancetheα+α+δ+β’fromRifR

E.coli

+

βfromRifSE.coliTheβsubunitisobviouslydeterminantofRifS

orRifR

ReconstitutionofRNApol.theα+α+δ+β’fromRifS

E.coli+βfromRifR

E.coli

ThereconstitutedRNApolymerasewasRifS

,regardlessoftheoriginoftheothersubunits

ThereconstitutedRNApolymerasewasRifR

,regardlessoftheoriginoftheothersubunits

Rifampin对RNApol

的抑制表现

●当I,Esite被pppXpYpZ(3Nts)填充，

Rif失去抑制效应

Rifampin是RNA合成起始的抑制剂

l

Rif与ATP/GTP对Isite的竞争

Rif紧密与β亚基结合→阻止

ATP/GTP对Isite

的填充

Zubay’sdiscovery(Lacoprontranscription)notranscriptionCAP-cAMPRifampicin+NtsRifampicin+Nts

AbsenceofCAP-cAMP

Rif.>NtsclosedpromotercomplexPresentofCAP-cAMP

polymerasebindingtightlytopromoter

Nts>Rif.openedpromotercomplex

CAP-cAMP

Nts>RifcompetitionNusAprotein;

★

69Kd,acidprotein★RNApol-attachingfactor

★

ImpelRNApol.pausinginterminatorandwaitingfor

Rho

factortostoptranscriptionofRNA★Afterinitiation→substitutingσ→NusA+coreE(antitermination

Nproteinutilizationsubstance)

原核生物转录的起始与延伸过程After2Nt30-50Nt/secG/C转录延宕PausinginTerm.σNusARhoPromoter与σfactor间的结合专效性

●决定了

strongpromoter&weakpromoter★不同启动子的-35，-10区序列间存在较为

保守的标准序列(标准启动子)

★启动子中-35,-10区序列的差异影响与

RNApol

中

σ因子的结合能力

σfactorisresponsibleforrecognitionofconsensussequenceofpromoterandonlyrequiredforinitiation.

TranscriptionalregulationbyalternativefactorinE.coliforstresscondition

When＞37℃;

Standardσ70

TTGACA16-18TATAAT-35-10Heatshockσ32

TTGAA-13-15CCCCAT-TWhen37℃;genesexpressed

inE.colibyRNApolwithσ70Morethen17proteinsareexpressedinE.colithroughtranscriptionbyRNApolusinganalternativeσ32,whichhaveownspecificpromoterconsensussequenceE.coli的热激响应是由一种可转变的σ-因子σ32(σH)控制，σ32取代σ70(σA)来介导RNA聚合酶与热激蛋白基因的启动子结合●

Manyphagesynthesizetheirownδfactorsinorderto“takeover”thehost’stranscriptionmachinerybysubstitutingthenormalhostδ(cascade)andalteringthepromoterspecificityofRNApolT4inE.coliandSPO1inB.Subtilis

Earlygenemiddlegeneslategenes

hostδ

phageδ28

phageδ-late

4.3.RNAtranscriptionpromotioninEukaryotes

2006年度诺贝尔化学奖授予美国科学家罗杰.科恩伯格，以表彰他对真核转录的分子基础所作的研究。

罗杰-科恩伯格的成就则是阐述了基因信息是如何从DNA被转录至信使RNA的。他制作了详细的结晶仪图片，描述了真核细胞转录的整个运转情况。可以看到新的RNA反转录酶是如何演变的，和数个转录过程中必需的其它分子的作用。使人们可以理解转录机制和转录是如何被管理的4.3.1.Biologicalstatebeforetranscriptionalstarting

l

细胞膜

l

Transcriptionalfactor(TF,transfactor)

RNApol

Cis-factor

l

染色体状态的调整

核小体

包装的解体

H3proteinouter

DNaseIS

外界信息的感知与传递细胞信号传递complex细胞核l

基本水平转录的

cis-factor

(promoterorbasicfactor)

Corepromoter(Capsite,TATAbox必需因子)

UPE(UpstreamPromoterElement)(CAATbox,GCbox)

Forhousekeepinggene(constitutiveexpression)

4.3.2.

与转录启动相关的cis-factors

l

特异诱导高效表达的cis-factor

EnhancerForluxurygene(inducibleexpression)

4.3.2.1.

Promoterforbasictranscription

(classIIrecognizedbyRNApolymeraseII)l

Capsite;initiationpoint(+1)Cappingm7GpppA/G70±AUG(inmRNA)1-4Kb-70-30+1

GCislandCAAC/TboxTATAbox

Cap

EnhancerUPEcorepromoter

Promoter(basicfactor)

InitiationregionPyPyANT/(A)PyPy

-globingene密集突变子转录效率upstreamsequenceTATABoxPromoterforbasictranscription

l

TATAbox/Hognessbox/Goldberg-Hognessbox(-30)RichATandrichGCflanked

richGC-TATAA(T)AA(T)richGC

8297938563(37)8350(37)1-4Kb-70-30+1

GCislandCAAC/Tbox

TATAbox

Cap

EnhancerUPEcorepromoter

Promoter(basicfactor)

TATAbox决定了转录起始点的选择

RNA聚合酶与TATAbox牢固结合之后才能开始转录

Promoterforbasictranscription

l

GCisland(Cnomethylated)andCAATbox(CAATboxUPE)(-70)

GCGCGGC(T)CAATCT

ResponsetoeffectoftranscriptionRange30bp±

1-4Kb-70-30+1

GCislandCAAC/Tbox

TATAboxCap

EnhancerUPEcorepromoter

Promoter(basicfactor)

CAATbox控制着转录起始的频率

Promoter的结构与功能

(Prok.E.coli

)

ABCGGACAATTATAAATG

HindIIIH4H2BH3H2AH1

HindIII

H22

HpaII

HinCII

HpaIH2A

-70(~60bp)-30(~60bp)+1(~80bp)

HpaII,HincII,HpaI,ligase

TaqI

XhoIPreludesequenceGrosschedl&MaxBernsteil(1980)SeaUrchininvivo

FirstidentifyingofPromoter’sfunctioninEuk.W.tH22△AH2A△BH2A△CH2A

H2BmRNAH2AmRNA1.01.90.220.24

Xenopus

leavis(frog)oocytes

GTPα32,purifyingRNA&electrophoresis△A(△B,△C)H2AfragmentofH22ofSeaUrchin

H2A/H2BGrosschedlandBimstiel

PNAS77(Mar1980)p.1434DeletionA

actuallystimulatedtranscription

DeletionC

hadlittleeffectontheamountoftranscription,butshiftedthestartsitedownstream,soashorterRNAappeared.DeletionB(includingtheTATAbox)causedadecreaseinamountoftranscriptionandtheappearanceofnewtranscriptsthatresultfromheterogeneousstartsitesA区的CAAT盒及两侧的GC岛控制着转录启动，缺失后转录上升TATAbox缺失，转录效率下降，而且不能正确选择转录起点起始位点缺失后，RNA转录酶需要在下游选择新的转录起点4.3.2.2.Cis-factorforinducibleexpression

SpecialboxArabidopsis

Ibox(GATAAG)5-20GboxPea

Hbox(CCTACC)5-20Gbox

modulHSE(HeatShockElement)GRE(

GlucocoricoidResponseElement)糖皮质激素应激元件MRE(MetalResponseElement)TSE(TissueSpecialElement)l

InducibleexpressedbyenvironmentIg

ATGGAAAT+Oct-2factor→B细胞表达GHATGAATAT+Pit-1factor→脑下垂体表达

Hotspotl

EnhancerEnhanceexpressionBasicexpressionPositionnotbefixedisolatedregionBi-directionalelementMono-directionalelementEnhancerPromoterNotforspecialgeneonlyforspecialgene★Enhancer与Promoter的比较SV40早期基因转录单位S

(Chambon)72bpregiondeletion

transcriptiongodown

intoupstreamofβ-globingene

Transcriptionup200X

Cell&tissuespecialityofEnhancereffectDistanceeffect(over2kb)Orientation-independent&

position-independent

Polarityeffect

BehaviorChambondiscoveredthefirstenhancerinthe5'-flankingregionoftheSV40earlygene.★Enhancer的结构与功能

Enhancer由两个以上的增强子成分(EnhancerElement)组成

EnhancerElement必需由两个紧密相连,具有间距效应的

增强子元

(Enhanson）组成

各个Enhanson(cis-factor)与激活蛋白(trans-factor)结合

增强特异性转录

促进转录的复合体

+UPE+corepromoter基本转录复合体Enhancer

Specifical

transc.Factor(tran-factor)

Specifical

transc.Factor(tran-factor)

……..++ActivationdomainofEnhancercomplex

ActivationdomainofEnhancercomplex

……..EnhancerElementEnhancerElement……..

(<100bp)

Enhanson(cis-factor)Enhanson(cis-factor)……..(<5bp)

En.Elem.En.ElemEn.Elem.-250-180

coreCTCIITCIsphII

sphI

Ap3Ap2Ap1e.g.SV40Enhancer(-179~-250)

远距离控制，无方向性

mRNA

+1GCCAATTATA

促进转录复合体基本转录复合体Loopingofpromoter-enhancerregionKustu,Echols1990PNAS87(July)促进转录的复合体+UPE+corepromoter

增强特异性转录

Enhancercomplex与近启动子的transcriptionalcomplex构型契合，而不与RNApolymerase结合

特化细胞内，具全部特异转录因子（trans-factor）才表现增强效应即增强子的组织特异性

增强子的复杂结构，以正控制方式防止基因表达的紊乱

Enhancer为两位点二元组织方式能利用有限的trans-factor提供更多基因的表达调控因子类型一位点的二元结构；A,Bfactor→AA,BB,AB,BA

两位点的二元结构；AA-AA,AA-BB,AA-AB,AA-BABB-BB,BB-AA,BB-AB,BB-BAAB-BB,AB-AA,AB-AB,BA-BA……

γ2b;agenethatencodesthelargersubunitofaparticularmouseantibody1983Cell33(July)p.719

1983Susunmu

TonegawafoundanenhancerwithinX2-x3ofγ2b

geneGillies1983Cell33(July)p.719

results:Thedeletionswithintheintronshouldhavenoeffectontheproteinproductbecause….Whythe∆1(180bpX3),∆2

(470bpX2-X3)causedadecreaseintheamountofgeneproductmade?

plasmidsAandB,theX2-X3

wasinsertedbackintotheintroninitsusuallocationintheforward(normal)orientation(A),orinthebackwardorientation(B);

plasmidsCandD,theX2-X3wasinsertedintoanotherXbalsite(X1)hundredsofbasepairsupstreamofthegeneintheforwardorientation(C),orinthebackwardorientation(D)Theenhancingelementintheγ2bgeneisorientation-andposition-independent.intronSilencer

canactatadistance(atleast1kbaway)tomodulatetranscriptionsomehowcausethechromatintocoilupintoacondensedInaccessibleInactiveformtherebypreventingtranscriptionofneighboringgenesSilencer沉默子:通过一段延伸的DNA区域影响染色质结构,从而调节(关闭)基因转录的DNA元件OgourneS.andAntalisT.M.1998.Transcriptionalcontrolandtheroleofsilencersintranscriptionalregulationineukaryotes.Biochem.J.331:1-14SIR(1-4)SilentInformationRegulatorNeg.controllingMatingtypegene-relatedinyeastsilentcassette

activecassette

silentcassetteHMLMATHMRaαα

/

athr4leuRegulationofHML/HMRsilencing

HML／HMR基因座两侧的DNA序列受SIR（沉默子结合蛋白）负控制

SIR指导异染色质结构的形成与保持

SIR与Histone

Deacetylation

有关

SIR与Histone结合，并扩展到相应的基因座，形成高密度的异染色质Insulator:(绝缘子)Elementsthatpreventthepassageofactivatingorinactivatingeffects能够阻断激活或失活效应通过的元件

具有两种功能:

Whenaninsulatorisplacedbetweenenhancer&promoter,itpreventstheenhancerfromactivatingthepromoterWhenaninsulatorisplacedbetweenactivegene&heterochromatin,itprovidesabarrierthatprotectsthegeneagainsttheinactivatingeffectthatspreadsfromtheheterochromatin4.3.3.与基本转录相关的T.F.Generaltranscriptionfactors

真核生物中RNApol的作用必须有其他相关转录蛋白在启动子处的先期结合才能启动转录EukaryoticRNApolymerasesareincapableofbindingbythemselvestotheirrespectivepromoters.Instead,theyrelyonproteinscalledtranscriptionfactorstoshowthemtheway.Suchfactorsaregroupedinto

Twoclasses:generaltranscriptionfactorsgene-specifictranscriptionfactors.

Twoclasses:

generaltranscription

factors(GTF)gene-specifictranscription

factors(STF)

Thegeneraltranscriptionfactors

GTFcanattracttheRNApolymerasestotheirrespectivepromoters,butonlytoaweakextent.Therefore,thesefactorscanstimulateonlyabasalleveloftranscription.4.3.3.与基本转录相关的T.F.Generaltranscriptionfactors(GTF)4.3.3.1GeneralT.F.forbasictranscription

l

TF(I,II,III)TranscriptionalFactorl

TBP(TATAboxBindingProtein)l

TAF(TBPAssociateFactor)l

RAP(RNApol.Associateprotein)真核生物中RNApol的作用必须有其他相关转录蛋白在启动子处的先期结合才能启动转录Cis-factor+

TIC(TranscriptionalInitiateComplex)转录起始复合物4.3.3.2.

RNApolymeraseinEukaryotes

RNApol.I,II,IIIL’with78%±homologousbetween3RNApol.I,II,IIIL’~β’ofprokaryoteRNApol.&L~β

(功能相似)IncludingL,L’subunit&7-12smallsubunits

RNApolII(B);

Forpre-mRNAtranscriptionL’maximumsubunit240kd&havespecificCOOH-end

namedCTD

(CarboxylTerminalDomain)onlyinRNApolII

CTD-end;7aarepeats&highfrequencyphosphorylationTyr—SerP—Pro—ThrP—SerP—Pro—SerP

Yeast26XDrosophila44Xrepeatunitof7aa/inCTDRat&Human52X

依CTD中，Ser,Thr

磷酸化与否，将L’分为3个Subforms

IIO

(240kd)

IIA(220kd)

IIB(180kd)

高度磷酸化CTDover-phosphorylated提高转录效率10XIIA蛋白酶水解Non-physiologicalform基本结合型

Initiallybindstopromoter

使RNApol易于离开Promoter转录延伸

RNApolII+>20TFIIs

逐级组装

TIC→转录启动

(TranscriptionalInitiationComplex)

TBP:neededforRNApolI,II,IIITFIID;Asortofproteincomplex(TBP&>8TAF)VeryhighconservedC-enddomainof180aaBindswithDNAinminorgroove&wildsitDeterminationinitiationstartingsite

ControlUPEeffectforbasictranscriptionTATA+1

Wildsminorgroove

TFIIATBPofD

pre-TIC

TFIIA;BindstoTFIIDEnhancesTFIIDbindingtoTATAboxStabilizingtheDNA-TFIIDcomplex

TFIIFRNApolIITFIIB

BasicTIC

TFIIB;OnceTFIIDhasboundtoDNATFIIBbindstoTBPofTFIIDandRNApol.II

TFIIBasabridgingfactor

allowing

recruitmentofRNApolII

toTICtogetherwithRAP30ofTFIIF

TFIIF;IncludingtwosubunitsofRAP30,RAP74BindingandrecruitingRNApolIItoassembleTIC

PromotingRNAelongationbyitshelicase

(ATPase)

bridgingfactorhelicase

(ATPase)TFIIE;AssociatewithTFIIHkinase

completeTIC

EHPhosphorylationofCTDofRNApolIIOTFIIH;Largecomplexmadeupof>5subunits

Helicaseactivity(ATPase,RAD25…)&kinase

activity

PhosphorylationofCTDofRNApolIIO

DNArepair

mRNA

Promoterclearance

TranscriptionstartingFEHBDATATA+1

Wildsminorgroove

TFIIATBPofD

pre-TIC

TFIIFRNApolIITFIIB

BasicTIC

conclusionmRNA

Promoterclearance

EHBDATranscriptionstartingcompleteTIC

EHGTF+TATAbox→basalleveltranscription

(a)ThepolymerasetranscribedtheDNAfromlefttoright,hasjustincorporatedanincorrectnucleotide(b)Thepolymerasebacktrackstotheleft,extrudingthe3'-endoftheRNA,withitsmis-incorporatednucleotide,outoftheactivesiteoftheenzyme.(c)Theribonucleaseactivityofthepolymeraseclipsoffthe3'-endoftheRNA,includingtheincorrectnucleotide.(d)Thepolymeraseresumestranscription.modelforproofreadingbyRNApolymeraseIIRNApolI(A);

Forpre-rRNA(—18s—5.8s—28s—)80-85%oftotalRNARNApolIII(C):16ormoresubunitsForpre-tRNA

5spre-rRNA,U6snRNA(smallnuclearRNA)ThegeneralT.F.arecapableofsponsoringonlyabasalleveltranscription.Toprovidetheneededextraboostintranscription,eukaryoticcellshaveadditional,gene-specifictranscriptionfactors

(activatorse.gGAL4).

4.3.3.3.transcriptionactivator(activator,STF)forgene-specificinducingtranscription

GAL4stimulatespreinitiationcomplexformationUPETATAa)

character

of

activator

l

多为变构蛋白，具有2或4个独立的功能结构域（domain）DNA-bindingdomainTranscriptionregulationdomainactivation

(orrepression)domainDNA-bindingdomainDimerizationdomain(insomedimerfactor)TranscriptionregulationdomainNuclearlocationsignal,