真核生物基因表达调控ppt课件

.,EukaryoticGeneExpressionandRegulation,第八章,真核基因表达调控,.,本章主要内容基因表达与调控的基本概念与原理转录水平的调控(transcriptionalregulation):DNAlevel(Genetic)Chromatinlevel(Epigenetic)转录后水平的调控(post-transcriptionalregulation)：RNAinterference（RNAi）Proteindegradation（Ubiquitin/proteasome）,.,第一节基本概念与原理,BasicConceptsandPrinciples,.,Genome(cellsrepertoireofDNA),Transcriptome(cellsrepertoireofRNAtranscripts),Proteome(cellsrepertoireofproteins),单个基因,单个细胞,中心法则,.,一、基因表达的概念,基因组(genome)一个细胞或病毒所携带的全部遗传信息或整套基因。,基因表达(geneexpression)基因经过转录、翻译，产生具有特异生物学功能的蛋白质分子或RNA分子的过程。,基因表达调控(generegulation,orregulationofgeneexpression)基因表达是受内源及外源信号调控的。,.,RegulationofGeneExpression,.,二、基因表达的时间性及空间性,（一）时间特异性,.,人体发育过程中不同类型-珠蛋白的含量变化,.,（二）空间特异性,基因表达伴随时间顺序所表现出的这种分布差异，实际上是由细胞在器官的分布决定的，所以空间特异性又称细胞或组织特异性(cellortissuespecificity)。,在个体生长全过程，某种基因产物在个体按不同组织空间顺序出现，称之为基因表达的空间特异性(spatialspecificity)。,.,BARD1isexpressedspecificallyintheapicaldomainsofArabidopsisinflorescence(A),ovules(B),anthers(C),andembryos(D).,InsuithybridizationA,B,C,D:antisenseBARD1probe;E:senseBARD1probeasanegativecontrol.,（朱玉贤第五章课件）,.,四种母源影响基因的mRNA和蛋白沿果蝇胚胎前-后轴分布的浓度变化图,protein,BICOID,NANOS,第十章（基因和发育）,mRNA,.,Facts,Identicalgenome:VirtuallyeverycellinanorganismcontainsacompletesetofgenesSpatialspecificity:ButtheyarenotallturnedonineverycellortissueTemporalspecificity:EachcellofanorganismexpressesadistinctivesubsetofgenesatdifferenttimeordevelopmentalstageTightregulation:Duringdevelopmentdifferentcellsexpressdifferentsetsofgenesinapreciselyregulatedfashion,.,三、基因表达的方式,按对刺激的反应性，基因表达的方式分为：,（一）组成性表达(constitutiveexpression),某些基因在一个个体的几乎所有细胞中持续表达，通常被称为管家基因(housekeepinggene)。,.,这类基因表达又称为组成性基因表达(constitutivegeneexpression)。,genesforessentialcellularstructuresandmetabolicpathways(e.g.rRNA,actin,tubulin)usuallyexpressedathighleveltheleveloftheirgeneexpressionmayvary,Housekeepinggenes,rRNA,actin,tubulinarecommonlyusedasloadingcontrolinRT-PCRorNorthernblot,.,（二）诱导和阻遏表达,在特定环境信号刺激下，相应的基因被激活，基因表达产物增加，这种基因称为可诱导基因(induciblegenes)。,如果基因对环境信号应答是被抑制，这种基因是可阻遏基因(repressiblegenes)。,基因表达调控大多数是对这些基因的转录和翻译速率的调节,从而导致其编码产物的水平发生改变，影响其功能。,.,四、基因表达调控的生物学意义,（一）维持细胞增殖、分化（二）维持个体生长、发育（三）适应环境变化,第九、十章（基因与疾病、基因与发育）将要讲到,.,1.Transcripts（转录本）beginandendbeyondthecodingregion(5UTRand3UTR),2.Theprimarytranscriptisprocessedby:5capping3formation/polyAsplicing,3.Maturetranscriptsaretransportedtothecytoplasmfortranslation,一般而言，基因表达调控主要是发生在基因转录水平上的调节，即：mRNA合成的多少。,transcription,.,五、基因转录调节基本要素（一）RNA聚合酶(RNAPolymerase)（二）特异DNA序列(cis-actingelements)（三）调节蛋白(trans-actingfactors),GeneexpressionregulationatthelevelofDNA(transcriptionalregulation)-highlysequence-dependent-variedregulationfordifferentgenes,.,cis-actingelements:promoters/regulatorysequencesofgenestrans-actingfactors:proteinsandRNAsthatbindcis-elementsandpromoteorrepressgeneexpression,.,(一)RNA聚合酶启动子、调节序列和调节蛋白通过DNA-蛋白质相互作用、蛋白质-蛋白质相互作用影响RNA聚合酶活性。RNAPolI:rRNA,相对活性50-70%RNAPolII:mRNA,相对活性20-40%RNAPolIII:tRNA,相对活性10%RNAPolIV:smallncRNA,相对活性?,.,真核生物基因组中含有可以调控自身基因表达活性的特异DNA序列，称为顺式作用元件(cis-actingelement)。顺式作用元件能够被转录调节蛋白特异识别和结合，从而影响基因表达活性。启动子(promoter)顺式作用元件又分增强子(enhancer)沉默子(silencer),（二）特异DNA序列,.,反式作用因子(trans-actingfactor)能直接或间接与顺式作用元件相互作用，进而调控基因转录的一类调节蛋白，统称为反式作用因子。按其功能不同，常有以下三类：基本转录因子:识别promoter元件转录调节因子:识别enhancer或silencer共调节因子：不能进行DNA-蛋白质相互作用,（三）真核基因的调节调节蛋白,.,RNA聚合酶在转录因子帮助下，形成的转录起始复合物,holoenzyme,1.基本转录因子(generaltranscriptionfactor,GTF)是指能够直接或间接与启动子核心序列TATA盒特异结合、并启动转录的一类调节蛋白。,TBP:TATA-boxbindingprotein,TFII:polIIassociatedTF,.,2.转录调节因子(transcriptionfactor,TF)这类调节蛋白能识别并结合转录起始点的上游序列和远端的增强子元件，通过DNA蛋白质相互作用而调节转录活性。决定不同基因的时间、空间特异性表达.转录激活因子(transcriptionalactivator)转录阻遏因子(transcriptionalrepressor)3.共调节因子(transcriptionalregulator/co-factor)首先与转录因子发生蛋白蛋白相互作用，进而影响它们的分子构象，以调节转录活性,本身无DNA结合活性。如果与转录激活因子有协同作用共激活因子；与转录阻遏因子有协同作用共阻遏因子。,.,常见转录因子的结构域(domain),DNA结合域（DNAbindingdomain)BasicAA(K/R)rich,positivelycharged,转录激活域(trans-activationdomain),TF,.,ZincFinger,bHLH,bZIP,Homeodomain,1）TF最常见的DNAbindingdomain,.,(1)锌指(zincfinger),常结合GCbox,Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X3-His,C-terminal:-helixbindingDNA,.,(2)碱性亮氨酸拉链bZIP,.,bHLH蛋白(basicHelix-Loop-Helix),(3)碱性螺旋-环-螺旋bHLH,.,2）TF常见的trans-activationdomain,.,(Activationdomainisinterchangeable),.,InteractionAssaysDesignofTwo-hybrid/Three-hybrid/etc,separablefunctionaldomains,Two-hybridassay(protein-protein),Tri-hybridassay(protein-RNA),.,1.RNApolymeraseII2.promoterandenhancers3.transcriptionfactors,Eukaryoticgeneexpressionisusuallycontrolledatthelevelofinitiationoftranscription.,真核基因转录起始的调控,.,Holoenzyme-asupramolecularcomplexcomprisingPolII,mostGTFs,andMediator/SrbcomplexInyeast,a2MDaholoenzyme+TBPsufficesfortranscription,OrderedAssemblyandPolIIHoloenzyme,TFIID,TFIID,one-step,multiple-step,.,TFIIBbindstoDNAandcontactsRNApolymeraseneartheRNAexitsiteandattheactivecenter,andorientsitonDNA.,+25bp,Q:prok-10bpvseuk-25bp?,SequentialAssembly,TBP:TATAbindingproteinTAFs:TBPassociatedfactors,BindingofTFIID(TBP+11TAFs,800KD)totheTATAboxisthefirststepininitiation.,.,CTD：RNAPolIIC-terminaldomain,CTDisanunusualextensionappendedtotheCterminusofthelargestsubunitofRNApolymeraseII.Itcomprisesfrom25to52tandemcopiesoftheconsensusrepeatheptadY1S2P3T4S5P6S7.S2andS5aremajorphosphorylationsites.CTDphosphorylationcausetheconversionofprolineisomerizationstates.PhosphorylationpatternsontheCTDrepeatsdeterminedifferentsetsofassociatedfactors,sothatprovideadynamicplatformtorecruitdifferentregulatorsofthetranscriptionapparatus.,Ineukaryoticcells,thetranscriptionofgenesisaccuratelyorchestratedbothspatiallyandtemporallybytheC-terminaldomainofRNApolymeraseII(CTD).,.,PIC:PhosphoS5isrequiredforassemblyofthePICandfacilitatesmRNAcappingviarecruitmentofcappingenzymes.Elongation:S5graduallybecomesdephosphorylated,whereasS2isphosphorylated.Terminating:PhosphoS2ensuresefficient3-RNAprocessingbytriggeringrecruitmentof3-RNAprocessingmachinery.Ending:CTDsarefreeofphosphategroups;non-phosphorylatedCTDsarerequiredforRNApolymeraseIItorecycleandbindapromoterforthenextcycleoftranscription.,S2&S5,thetriggerfortranscriptionalprocessmodulation,.,.,FactorsinvolvedingeneexpressionincludeRNApolymeraseandthebasalapparatus,activatorsthatbinddirectlyto,co-activatorsthatbindtobothactivatorsandthebasalapparatus,andregulatorsthatactonchromatinstructure(chromatinremodelingcomplex).,ManyTranscriptionalActivators,i.e.CAATGC-box,.,Neartheinitiationsite,Alittlefaraway,.,SP1stimulatestranscriptioninpresenceofTAFII110,GCboxesboundbyDNAbindingproteinSP1SP1recruitsTFIIDbybindingTAFII110Partiallyreconstitutedcomplex(TBPand3TAFs)inadditiontootherGTFs,PolIIleadstohighlevelsoftranscription,SV40earlypromoter,Near,.,Mediatorcomplexistargetedbyanactivator,Mediatorisastablecomplexcontainingseveralproteins(20-50)MediatorbindstotheRNApolIIandtranscriptionfactors(activatorsorrepressors)andmediatestheregulatorysignalstopolII,(中介复合体）,Far,.,Whatisthemechanismofactivation?,Twomodels:Tetheringholoenzyme(recruitment)Activatingholoenzyme(allosteric),.,Infavorofrecruitmentmodel,（勾引模型）,.,tatproteinofHIVcanstimulatetranscriptioninitiationwithoutbindingDNAatallTheactivatingdomainofthetatproteincanstimulatetranscriptionifitistetheredinthevicinityofpromoterbybindingtotheRNAproduct(tarsequence)ofapreviousroundoftranscription.,tar,tat,.,DNA-bindingdomainistobringtheactivationdomainintothevicinityofthestartpoint.Andactivationisindependentofthemeansoftethering.,wecanthinkofDNA-binding(orRNA-bindinginthecaseoftat)domainasprovidingatetheringfunction,whosemainpurposeistoensurethattheactivationdomainisinthevicinityoftheinitiationcomplex.Thenotionoftetheringisamoregeneralideathatinitiationrequiresahighconcentrationoftranscriptionfactorsinthevicinityofthepromoter.Thismaybeachievedwhenactivatorsbindtoenhancers,upstreampromoterelements,orinanextremecasebytetheringtoanewly-madeRNAproduct.,.,总结所有激活因子的共性：识别靶位点（启动子、增强子）的特异性由DNA结合域决定。DNA结合域将转录激活域带到基础转录区域附近。直接作用的激活因子具有DNA结合域和转录激活域。没有转录激活域的激活因子可能与具有转录激活域的共激活因子一起行使功能。基础转录区域中许多元件是（共）激活因子的靶位点RNA聚合酶可以和多种不同的转录因子相互作用，形成全酶复合物行使功能。,.,SynergyHighlevelsoftranscriptioninducedbymultiplefactors,Transcriptionfactorscanenhancetranscriptioninanon-linearmannerSynergisiticactivationoccursduetomultiplecontactswiththemachineryMultiplecopiesofthesameactivatoralsoinducesynergisticactivation,.,Interferonenhancer,EnhancersoftenhavebindingsitesforseveraltranscriptionfactorsTranscriptionfactorscanbindcooperativelyatadjacentsitesArchitecturalfactors(withnoregulatorydomains,i.e.HMG1)canassistassemblyRemarkablyincreasebindingaffinityforbothDNAandmachinery,.,HMG1,肩并肩、手挽手，根基稳、魅力足,香肩并立、玉指紧扣，脚如磐石、面若桃花,.,Onepossiblestrategy:,-Looping-,Cohesinshelptostabilizeenhancer-promoterinteractions,Howdoenhancersactindependentofdistanceandorientation?,.,Anenhancermayfunctionbybringingproteinsintothevicinityofthepromoter.AnenhancerdoesnotactonapromoterattheoppositeendofalonglinearDNA,butbecomeseffectivewhentheDNAisjoinedintoacirclebyaproteinbridge.AnenhancerandpromoteronseparatecircularDNAsdonotinteract,butcaninteractwhenthetwomoleculesarecatenated.,Twoexperimentssupporttheloopingmodel,-Theessentialroleoftheenhanceristoincreasetheconcentrationofactivatorinthevicinityofthepromoter-,.,Receptorsformanysteroidandthyroidhormoneshaveasimilarorganization,withanindividualN-terminalregion,conservedDNA-bindingregion,andaC-terminalhormone-bindingregion,Steroidreceptorsaretranscriptionfactors,ZincfingerTF,.,Glucocorticoidsregulategenetranscriptionbycausingtheirreceptortotransportintothenucleusandbindtoanenhancerwhoseactionisneededforpromoterfunction.,ActivationofGlucocorticoidReceptor(GR),Nuclearshuttling,.,利用GR的特性构建可诱导表达融合蛋白系统,Dexamethasone（DEX）:地塞米松,氟美松(抗炎药)，合成的一种糖皮质激素；通过分子克隆的方法将GR和要研究的核蛋白X构建成融合蛋白，转基因到酵母、动物细胞或者植物中；不施加外源DEX时，融合蛋白与HSP90形成复合物，由于构象和空间位阻等原因，融合蛋白存在于胞质中，不能定位到细胞核；添加DEX时，DEX扩散入胞与GR相结合，融合蛋白改变构象后核定位信号暴露，即可入核行使功能；用以研究核蛋白的功能（包括转录因子）。,.,ActivationTaggingapproachinplants,Planttransformation,mutantsscreeninglocateT-DNAinsertionsiteinArabidopsisgenome(how?)identifytherightgeneconferringmutantphenotype(how?),构建T-DNA序列，其中包括4倍重复的CaMV的35S增强字序列，435S元件可以大大增强相邻基因的转录；通过转基因的方法将T-DNA片段整合到植物基因组中；植物基因组上与T-DNA插入位点相近的基因表达量增高，即得到这个基因gain-of-function的转基因植物；,.,XVELexA的DNA结合域(X)VP16转录激活域(V)humanestrogenreceptor调控域(E)NPTII；转基因筛选标记OLexA-46：LexA操纵子CaMV35S基本启动子（而非增强子）,Achemical-inducibleactivationtaggingvectorpER16inplants,RB-LB:T-DNAfragment(可以插入到基因组中),其工作原理为：在G10-90启动子控制下，XVE融合转录因子组成型表达；当加入雌激素，雌激素和受体调控域（E）结合，导致XVE融合蛋白构象发生变化，并由细胞质转移进入核内；在细胞核内，XVE中的LexADNA结合域（X）特异识别LexA操纵子区（OLexA），VP16的转录激活域（V）激活LB旁边的基因高水平表达。,.,EnhancerTrap,Enhancertrap的质粒包含一个报告基因（lacZ）和基本启动子，这段启动子不足以启动报告基因的表达，但是对增强子非常敏感。将这样的质粒整合到基因组中，如