真核生物轉(zhuǎn)錄機(jī)理與調(diào)控匯總

MolecularBiologyChaper4TranscriptioninEukaryotes:MechanismandRegulationContent4.1EukaryoticRNApolymerases4.2EukaryoticPromoters4.3GeneralTranscriptionFactorsandInitiation4.4SpecificTranscriptionFactorsandTranscriptionalRegulation4.5Structuresofspecifictranscriptionfactors4.6ExperimentsTranscriptioninEukaryotes:MechanismandRegulationProkaryoteEukaryoteTranscriptioninprokaryotesTranscriptioninEukaryotes:MechanismandRegulationEukaryoticRNApolymeraseIIandtranscriptionfactorsSpecifictranscriptionfactorGeneraltranscriptionfactorsGeneraltranscriptionfactorsTranscriptioninEukaryotes:MechanismandRegulation表4.1表4.1核仁核基質(zhì)核基質(zhì)4.1EukaryoticRNApolymerasesLocationsofRNApolymeraseNucleolus(RNApolymeraseI)Nucleoplasm(RNApolymeraseIIandIII)RNApolymeraseIrRNAprecursorProcessing5.8SrRNA18SrRNA28SrRNARNApolymeraseIRNApolymeraseIIRNApolymeraseIIRNAprecursorProcessingsnRNAsTranslationProteinmRNAHoloenzyme:

Coreenzyme(β’,β&α)+SubunitσRNApolII:

Coresubunits(1,2&3)+Subunits4-12RNApolymeraseIIRpb1、Rpb2和Rpb3稱(chēng)為‘核心亞基’RNApolymeraseII蟹鉗RNApolymeraseIIIRNApolymeraseIIIRNAprecursorProcessing5SrRNA4.5StRNAU6snRNATranscriptioninEukaryotesTranscripitionofrRNAgeneTranscripitionoftRNAand5SrRNAgeneTranscripitionofmRNAandsnRNAgenes

RNAPolIRNAPolIIRNAPolIIImRNAThreetypesofRNApolymeraseanditstranscriptinEukaryotes4.2EukayoticPromoters4.2EukayoticPromotersProkaryoticpromoterEukaryoticpromoter4.2EukaryoticPromotersTATA框起始子TFIIB識(shí)別元件上游啟動(dòng)子元件下游啟動(dòng)子元件“IwouldliketohaveTATAboxandInitiatorasmypromoter.Whatwouldyoulike?”“Well,IpreferBREandDPE.”EukaryoticpromoterTATAAA-10boxTATAATTATAboxTATAboxSpecializedgenes+Housekeepinggenes–Genesofdevelopment–

TATA框最常出現(xiàn)在特殊的基因里GCGAT

DPE:DownstreamPromoterElementPyPyANPyPyTA驅(qū)動(dòng)基礎(chǔ)轉(zhuǎn)錄

Initiator/起始子drivebasictranscriptionCGCCGGGCCABRE:TFIIB

RecognitionElement原核生物RNA聚合酶前起始復(fù)合體真核生物RNApolymeraseII:“Ineedhelpfromotherproteins.”4.3GeneralTranscriptionFactorsandInitiation4.3.1TFIIDTBP:TATA-BindingProteinShow3DstructureofTBPTBP與TATA框結(jié)合的圖片TAFIIs:TBP-AssociatedFactors

IITAFIIs:1)BindingtoDPEorInr結(jié)合到核心啟動(dòng)子元件上而不是結(jié)合到TATA框上去促進(jìn)轉(zhuǎn)錄。TAFIIs:2)BindingtoActivator與特異轉(zhuǎn)錄因子結(jié)合以增強(qiáng)或抑制轉(zhuǎn)錄4.3.2OtherTFIIs TFIID（TBP）結(jié)合TATAbox↓TFIIA和TFIIB結(jié)合BRE↓RNAPolII-TFIIFisrecruited↓TFIIEandTFIIHbindupstreamofPolIItoformthepre-initiationcomplex↓PromotermeltingusingenergyfromATPhydrolysisbyTFIIH)↓PromoterescapesafterthephosphorylationoftheCTDtail4.3.3GeneralTranscriptionfactors

forRNAPolymeraseIandIII

RNA聚合酶I和III的通用轉(zhuǎn)錄因子（1）GeneralTFsofRNApolymeraseISL1結(jié)合到核心元件上并幫助召集RNA聚合酶I到啟動(dòng)子位置。UBF結(jié)合到上游啟動(dòng)子元件幫助SL1結(jié)合到核心元件上。I類(lèi)啟動(dòng)子核心元件上游啟動(dòng)子元件RNAPolIpromotersInitiationofpolⅠgene’stranscriptionUBFUBFSL1POLⅠNOTE:UBF----upstreambindingfactorSL1----selectivityfactor

5SrRNApromotertRNApromoter(2)RNApolymeraseIII的通用轉(zhuǎn)錄因子

①ThreetypesofpolIIIpromoters②InitiationoftranscriptionoftRNApromoter

GeneralTFsofRNApolymeraseIII③Initiationoftranscriptionof5SRNApromoter①EukaryotictRNApromoter②Eukaryotic5SrRNApromoterAboxBboxTATAbox+1-30-23AboxCboxTATAbox+1-30-23Promoters(啟動(dòng)子)ofRNApol.IIITFCTFBTFATFCTFBTFⅢC+1內(nèi)啟動(dòng)子Abox+50~+65Cbox+81~+995srRNAgeneTFⅢBPolⅢTFⅢAPolⅢpromoter&transcriptioninitiation內(nèi)啟動(dòng)子AboxBboxtRNAgeneD-loopTψC-loop+1TFⅢCTFⅢBPolⅢ4.4SpecificTranscriptionFactorsandTranscriptionalRegulation

特異轉(zhuǎn)錄因子與轉(zhuǎn)錄調(diào)控Specifictranscriptionfactor真核生物采用特異轉(zhuǎn)錄因子的蛋白質(zhì)來(lái)改變轉(zhuǎn)錄速率Specifictranscriptionfactors

特異性轉(zhuǎn)錄因子Specifictranscriptionfactors:

Eukaryoticfactorsthatchangetherateoftranscriptionoftargetgenes.特異性轉(zhuǎn)錄因子：改變目標(biāo)基因轉(zhuǎn)錄速率的的真核生物因子。

Factors（因子）Proteins（蛋白質(zhì)）Elements（元件）DNAsegments（DNA區(qū)段）Q→Whatarethetwotypesofspecifictranscriptionfactors?Howcantheyaffectinitiationoftranscription?

特異轉(zhuǎn)錄因子分哪兩種類(lèi)型？

它們是如何影響轉(zhuǎn)錄起始的？SpecifictranscriptionfactorGeneraltranscriptionfactorsGeneraltranscriptionfactorsSpecificTranscriptionFactorsandTranscriptionalRegulation

特異轉(zhuǎn)錄因子與轉(zhuǎn)錄調(diào)控4.4.1Activators/激活蛋白4.4.2Repressors/阻遏蛋白Specifictranscriptionfactorsthatenhancetherateoftranscriptionarecalledactivators.Helprecruitthepre-initiationcomplex.Helporganizethesubunitsofthepre-initiationcomplex,andmaintaincohesionofthecomplex.AttractenzymestoloosenDNAfromproteins,freeingimportantregionslikethepromoter.4.4.1Activators/激活蛋白（1）概念（2）調(diào)控機(jī)理①Helprecruitthepre-initiationcomplex②Helporganizethesubunitsofthepre-initiationcomplex③AttractenzymestoloosenDNASpecifictranscriptionfactorsthatpreventthetranscriptionofagenearecalledrepressors.能阻礙基因轉(zhuǎn)錄的特異轉(zhuǎn)錄因子稱(chēng)為阻遏蛋白。Blockingtheactivityofactivators.Blocking

accessofthepre-initiationcomplextothepromoter.RecruitingenzymesthattightenDNAaroundproteins.4.4.2Repressors/阻遏蛋白（1）概念（2）調(diào)控機(jī)理①Blockingtheactivityofactivators②Blockingaccessofthepre-initiationcomplextothepromoter.③RecruitingenzymesthattightenDNAaroundproteins順式作用元件（cis-actingelement）指與結(jié)構(gòu)基因表達(dá)調(diào)控相關(guān)、能夠被基因調(diào)控蛋白特異性識(shí)別和結(jié)合的特異DNA序列。包括啟動(dòng)子、上游啟動(dòng)子元件、增強(qiáng)子等。指能直接或間接地識(shí)別或結(jié)合在各類(lèi)順式作用元件核心序列上參與調(diào)控靶基因轉(zhuǎn)錄效率的蛋白質(zhì)。

反式作用因子(trans-actingfactor)4.4.3EnhancersandSilencers

增強(qiáng)子和沉默子順式作用元件能與特異性轉(zhuǎn)錄因子結(jié)合，以決定轉(zhuǎn)錄的起始位點(diǎn)、轉(zhuǎn)錄效率及轉(zhuǎn)錄的時(shí)空特異性EnhancersandSilencersTranscriptionisactivatedEnhancerTranscriptionisrepressedSilencerRepressorActivator增強(qiáng)子是一類(lèi)正調(diào)控順式作用元件，能夠從轉(zhuǎn)錄起始點(diǎn)的上游或下游數(shù)千個(gè)堿基處來(lái)激活轉(zhuǎn)錄，使和它連鎖的基因轉(zhuǎn)錄頻率明顯增加的DNA序列。

（1）增強(qiáng)子（Enhancers

）TranscriptionisactivatedEnhancerActivatorDNAlooping/DNA成環(huán)SeaUrchinEndo16gene

（海膽Endo16基因）Aredseaurchin紅海膽海膽Endo16基因的多增強(qiáng)子調(diào)控模式（2）沉默子（Silencers

）能夠同反式因子結(jié)合從而阻斷增強(qiáng)子及反式激活因子的作用，并最終抑制該基因的轉(zhuǎn)錄活性。TranscriptionisrepressedSilencerRepressorQuestionsQ1

→WhatkindsofDNA-bindingmotifsdoeukaryoticproteinshave?

真核生物具有什么樣的DNA結(jié)合基序?Q2

→HowdidweknowproductsofvariousRNApolymerases?

我們是如何知曉不同RNA聚合酶的產(chǎn)物的？Q3

→Areproteinmodulesexchange-able?

蛋白質(zhì)模塊可以互換嗎？??ProkaryoticEukaryotic4.5特異轉(zhuǎn)錄因子的結(jié)構(gòu)大溝A≠TG≠C小溝A≈TG≈C大溝VS小溝轉(zhuǎn)錄因子的結(jié)構(gòu)DNA結(jié)合結(jié)構(gòu)域轉(zhuǎn)錄活化結(jié)構(gòu)域4.5.1DNA-BindingMotifsinProkaryotes原核生物DNA結(jié)合基序RecognitionhelixThehelix-turn-helix(HTH)motifHelix-turn-helix:AcommonDNA-bindingmotifinprokaryoticproteins.Consistsoftwoα-helicesconnectedbyashortpeptideturn.螺旋-轉(zhuǎn)角-螺旋：原核生物中常見(jiàn)的DNA結(jié)合基序。由兩個(gè)α-螺旋通過(guò)一個(gè)肽轉(zhuǎn)角連接在一起。)例子：lac阻遏蛋白Show3Dstructureofthelacrepressor例子：Trp阻遏蛋白TrpTrprepressorDNAE.coliOperator例子：Trp阻遏蛋白Show3Dstructureofthetrprepressor4.5.1DNA-BindingMotifsineukaryote 真核生物DNA結(jié)合基序(1)Thehomeodomain

同源異型域(2)Zincfingermotif

鋅指基序(3)Leucinezipper

亮氨酸拉鏈(4)HLHmotif

螺旋-環(huán)-螺旋基序?Eukaryotic(1)Thehomeodomain/同源異型域SimilartoHTHofprokaryotesHomeodomain:DNA-bindingdomainfoundinmanyeukaryoticproteinsthatareinvolvedinorganismaldevelopment.同源異形域：在許多真核生物中發(fā)現(xiàn)的DNA結(jié)合域，在生物發(fā)育中起作用。由短的肽鏈轉(zhuǎn)角連接在一起的三個(gè)α螺旋組成。其中兩個(gè)螺旋在一起看起來(lái)與HTH基序非常相似。SimilartoHTHofprokaryotes(1)Thehomeodomain/同源異型域與DNA大溝發(fā)生特異性接觸支撐作用肽鏈延伸段，深入到DNA的小溝①同源異型基因(Homeoticgenes)同源異型基因是指一類(lèi)含有同源框的基因。在胚胎發(fā)育中的表達(dá)水平對(duì)于組織和器官的形成具有重要的調(diào)控作用。該類(lèi)基因的突變，就會(huì)在胚胎發(fā)育過(guò)程中導(dǎo)致某一器官異位生長(zhǎng)，即本來(lái)應(yīng)該形成的正常結(jié)構(gòu)被其他器官取代了。①同源異型基因(Homeoticgenes)廣泛存在于從酵母到人類(lèi)的各種真核生物中的基因，其特點(diǎn)是基因中存在共同的180bp的DNA片段，編碼高度同源的60個(gè)氨基酸，這個(gè)共同的180bpDNA片段稱(chēng)為同源異型框（homeobox）,含有同源異型框的基因稱(chēng)之為同源異型基因。如果蠅的HOM基因，動(dòng)物和人類(lèi)的Hox基因。在胚胎發(fā)育中起著類(lèi)似萬(wàn)能開(kāi)關(guān)的作用，保證生物在正常的位置發(fā)育出正常形態(tài)的軀干、肢體、頭顱等器官。②同源異型蛋白和同源異型域（homeodomain）由同源異形框基因編碼的蛋白質(zhì)稱(chēng)為同源異型蛋白。同源框由180個(gè)堿基組成，編碼一段稱(chēng)為同源異形域的特定氨基酸序列（60aminoacids），稱(chēng)為同源異型域。MutationofhomeoticgenesHomeoticgenes觸角足畸形突變同源異形基因負(fù)責(zé)在正確的位置發(fā)育出正確的器官Show3Dstructureofhomeodomain(2)Zincfingermotif/鋅指基序Zincfingers:DNAbindingdomainsthatareelongated,withashapethatissomewhatlikeafinger.Theshapeofthedomainiscoordinatedbyoneormorezincatoms.鋅指結(jié)構(gòu)：伸展的、形狀有些像手指的DNA結(jié)合域。這一形狀由一個(gè)或多個(gè)鋅原子進(jìn)行協(xié)調(diào)。OneproteinhasmultiplezincfingersFinger1Finger2Finger3Whysomany?(3)Leucinezipper/亮氨酸拉鏈Leucinezipper:ADNA-bindingdomainformedbytwoα-helices,usuallyfromtwodifferentproteinsthatcometogetherattheDNA.Thehelicesareheldtoeachotherbyhydrophobicinteraction,asbotharelinedwithleucineaminoacids.亮氨酸拉鏈：一種由兩個(gè)α螺旋組成的DNA結(jié)合域形式，通常由一起來(lái)到目標(biāo)DNA的兩種不同蛋白質(zhì)組成。兩個(gè)螺旋通過(guò)疏水相互作用而保持在一起，因?yàn)閮烧叨季哂信挪汲梢粭l線(xiàn)的亮氨酸。Show3Dstructureofleucinezipper這類(lèi)蛋白質(zhì)的DNA結(jié)合結(jié)構(gòu)域?qū)嶋H是以堿性區(qū)和亮氨酸拉鏈結(jié)構(gòu)域整體作為基礎(chǔ)的。Homodimerization/同源二聚化作用LeucineHomodimerization:

Formationofdimersbetweentwoidenticalsubunits.同源二聚化作用：兩個(gè)相同亞基之間形成二聚體的過(guò)程。Heterodimerization/異源二聚化作用Heterodimerization:

Formationofdimersbetweentwodifferentsubunits.異源二聚化作用：兩個(gè)不同亞基之間形成二聚體的過(guò)程。異源二聚體能夠識(shí)別不同的DNA序列，這一特性能夠使較少種類(lèi)的蛋白產(chǎn)生出更多種類(lèi)的DNA結(jié)合域。思考題：你克隆到的一個(gè)蛋白質(zhì)，序列如下：

001

MESADFYEVEPRPPMSSHLQSPPHAPSNARLWLSPGRGPRAAPSPTCRPGAAGRICEHET

061

SIDISAYIDPAAFNDEFLADLFQHSRQQEKAKAAAGPAGGGGDFDYPGAPAGPGGAVMSA

121GAHGPPPGYGCAAAGYLDGRLEPLYERVGAPALRPLVIKQEPREEDEAKQLALAGLFPYQ

181PPPPPPPPHPHASPAHLAAPHLQFQIAHCGPTPPPTPVPSPHAAPALGAAGLPGPGSALK

241GLAGAHPDLRTGGGGGGSGAGAGKAKKSVDRDKAKQRNVELTQQKVELTSDNDRLRKRVE

301QLSREADTLRGIFRQLPESSVKLAMATARLARGCGTLAGRPPGLWRPRGWFRVAGSGCPG

361RASQD

根據(jù)已知的研究結(jié)果表明它是一個(gè)轉(zhuǎn)錄因子。從序列計(jì)算得到的分子量為40kDa，但從蛋白質(zhì)用離心沉降方法得到的分子量為80kDa，你認(rèn)為最可能的原因是什么?請(qǐng)根據(jù)你提出的假說(shuō)設(shè)計(jì)試驗(yàn)，并預(yù)測(cè)如果假說(shuō)正確時(shí)的實(shí)驗(yàn)結(jié)果。根掘氨基酸的序列，你認(rèn)為什么特點(diǎn)使得這個(gè)蛋白質(zhì)發(fā)生了你所提出的假說(shuō)中的現(xiàn)象，并設(shè)計(jì)實(shí)驗(yàn)加以驗(yàn)證。(4)Helix-loop-helixmotif/螺旋-環(huán)-螺旋基序Thehelix-loop-helix(HLH)motifHelix-loop-helix/螺旋-環(huán)-螺旋Thehelix-loop-helix(HLH)motifHelix-loop-helix:ADNA-bindingdomaininproteins.Eachhalfofthedomainconsistsoftwoα-helicesconnectedbyapeptideloop.螺旋-環(huán)-螺旋：蛋白質(zhì)的一種DNA結(jié)合域。該結(jié)合域的每一半由兩個(gè)α-螺旋通過(guò)一個(gè)肽環(huán)連接在一起。HLH基序由通過(guò)一個(gè)環(huán)連接在一起的兩個(gè)α螺旋組成。它也像一把鉗子一樣抓住DNA。HLH

versus

HTHThehelix-loop-helix(HLH)motifThehelix-turn-helix(HTH)motifShow3DstructureofHLH4.6Experiments /實(shí)驗(yàn)研究4.6.1RNApolymerasetargets

RNA聚合酶的目標(biāo)4.6.2ModularityofSpecificTranscriptionFactors

特異轉(zhuǎn)錄因子的模塊化4.6.1RNApolymerasetargets

RNA聚合酶的目標(biāo)一開(kāi)始是怎么知道的呢？α-amanitin/α鵝膏蕈堿RNApolymeraseIRNApolymeraseIIRNApolymeraseIIIrRNAprecursorHighconcentrationofα-amanitinRNApolymeraseIproducesrRNAprecursors.為什么α鵝膏蕈堿對(duì)人類(lèi)有這么大的毒性？RNApolymeraseIRNApolymeraseIIRNApolymeraseIIIrRNAprecursortRNA,5SrRNA…Lowconcentrationofα-amanitinRNApolymeraseIIIproducestRNA,5SrRNAandothersnRNAs.為什么α鵝膏蕈堿對(duì)人類(lèi)有這么大的毒性？ProductofRNApolymeraseII

RNA聚合酶II的產(chǎn)物NomRNARNApolymeraseIIdoesnotworkRNApolymeraseIIisresponsibleformakingmRNA.4.6.2ModularityofSpecificTranscriptionFactors

特異轉(zhuǎn)錄因子的模塊化Modularity:referstothecompositionofaproteinwithseparatestructuralmodules.模塊化：指蛋白質(zhì)由不同的結(jié)構(gòu)模塊組成。Modules:

Proteindomainsthatarefoundinmanyproteinsandarewellconservedinevolution.模塊：在許多蛋白質(zhì)中存在并且在進(jìn)化中相當(dāng)保守的蛋白質(zhì)域。特異轉(zhuǎn)錄因子通常是模塊化的，含有一個(gè)DNA結(jié)合模塊和一個(gè)分開(kāi)的激活模塊。Q

→Areproteinmodulesexchange-able?蛋白質(zhì)模塊可以互換嗎？4.6.2

ModularityofSpecificTranscriptionFactors

特異轉(zhuǎn)錄因子的模塊化Moduleexchange模塊交換模塊化轉(zhuǎn)錄和/或翻譯產(chǎn)物可以很容易地被檢測(cè)出來(lái)的基因Thereportergenes/報(bào)告基因Braincancermodels

HumanU-87-RFPgliomagrowinginbrainofnudemouse.腦瘤模型：生長(zhǎng)在裸鼠腦中的人神經(jīng)膠質(zhì)瘤RFPGFP報(bào)告基因綠色熒光蛋白ThemodularityofGal4

(轉(zhuǎn)錄激活蛋白Gal4)模塊化upstreamactivationsequenceThemodularityofGal4

(轉(zhuǎn)錄激活蛋白Gal4)模塊化ThemodularityofGal4

(轉(zhuǎn)錄激活蛋白Gal4)模塊化YeastOneHybridSystem4.1真核生物RNA聚合酶4.2真核生物啟動(dòng)子4.3通用轉(zhuǎn)錄因子與轉(zhuǎn)錄起始4.4特異轉(zhuǎn)錄因子與轉(zhuǎn)錄調(diào)控4.5特異轉(zhuǎn)錄因子的結(jié)構(gòu)4.6實(shí)驗(yàn)研究 主要內(nèi)容4.5.2DNA-BindingMotifsineukaryote 真核生物DNA結(jié)合基序1.Homeodomain

同源異型域2.Zincfingermotif

鋅指基序3.Leucinezipper

亮氨酸拉鏈HLHmotif

螺旋-環(huán)-螺旋基序?EukaryoticThehomeodomain/同源異型域SimilartoHTHofprokaryotesHomeodomain:DNA-bindingdomainfoundinmanyeukaryoticproteinsthatareinvolvedinorganismaldevelopment.同源異形域：在許多真核生物中發(fā)現(xiàn)的DNA結(jié)合域，在生物發(fā)育中起作用。由短的肽鏈轉(zhuǎn)角連接在一起的三個(gè)α螺旋組成。其中兩個(gè)螺旋在一起看起來(lái)與HTH基序非常相似。Zincfingermotif/鋅指基序Zincfingers:DNAbindingdomainsthatareelongated,with