《英汉对照分子生物学导论》课件 王勇 Chapter 3 Transcription、Chapter 4 Transcription in Eukaryotes Mechanism and Regulation

Chapter3/第3章Chapter3 TranscriptioninProkaryotes:MechanismandRegulation3.1WhyUseanRNAIntermediate?3.2MechanismofTranscription3.3RegulationofGeneExpressioninProkaryotes3.4Experiments第3章原核生物转录：机理与调控3.1为什么使用RNA

作为中间物？3.2转录机理3.3原核生物基因表达调控3.4实验研究3.1WhyUseanRNAintermediate?

3.1为什么使用RNA作为中间物？ChromosomesarelargeandcomplexLowefficiency!DNAProteinRibosomeDNAmustbestableeOnemRNAmanyproteinsMoreopportunitiesforregulationChapter3Chapter43.2MechanismofTranscription

3.2转录机理3.2.1Promoters /启动子−10boxand−35boxCCAGGCTTTACACTT---CGTATGTTGTGTGGAATTCTTTTTGATGCAATT---ACTATAATAGACAGGGTAGGCGGTGTTGACATA---GTGATACTGAGCACATCA

-35box-10box

+1Consensussequence/共有序列CCAGGCTTTACACTT---CGTATGTTGTGTGGAATTCTTTTTGATGCAATT---ACTATAATAGACAGGGTAGGCGGTGTTGACATA---GTGATACTGAGCACATCA

-35box-10box

+1TATAATTTGACAConsensussequencesConsensussequence:ageneralizedsequencefromwhichmostactualsequencesdifferverylittleornotatall.（共有序列：一种普遍的序列，大多数实际的序列与它相差很小或完全相同。）3.2.2RNAPolymerase/RNA聚合酶Coreenzymevs.HoloenzymeHoloenzymestartstranscriptionatpromotersCoreenzymestartstranscriptionatrandomCatalyticareaofRNApolymerase3.2.3TranscriptionMechanism

3.2.3转录机理Initiation/起始

Elongation/延伸

Termination/终止1.Initiation/起始Step1Step2Initiation/起始Step3Step42.Elongation/延伸TranscriptionbubbleThepolymerizationreaction

聚合反应RNAsynthesis:5’→3’Proofreading/校正UCRemoveUAddCNowshowing

TranscriptionFile:biophoto7\Life\Transcription.(5’)3.Termination/终止Intrinsictermination内在型终止ρ-dependentterminationρ依赖型终止Intrinsictermination/内在型终止

Basepair/碱基对ρ-dependenttermination/ρ依赖型终止

RhoproteinsX-RayCrystalStructureOfRhoprotein[MMDBID:24381]

Nowshowing

BacterialTranscriptionFile:biophoto7\Genetics:AConceptualApproach\Bacterialtranscription(6’).3.3Regulationofgeneexpression

inprokaryotes

3.3原核生物基因表达调控3.3.1CoordinateRegulation3.3.2TheLacOperon3.3.3TheTrpOperon3.3.4Ara&GalOperons3.3.1协同调控3.3.2乳糖操纵子3.3.3色氨酸操纵子3.3.4阿拉伯糖与半乳糖操纵子Regulationofgeneexpression

基因表达调控ConditionARegulationofgeneexpression

基因表达调控ConditionB3.3.1CoordinateRegulation

协同调控Operator操纵基因PolycistronicmRNA

多顺反子mRNAPolycistronicmRNATranslationProtein1Protein2Protein33.3.2ThelacOperon

/乳糖操纵子Thelacoperon1.TheConditionsoflactoseMetabolism

1.

乳糖代谢的条件E.coli:“Thisismyfavorite!”乳糖半乳糖葡萄糖Howtouselactose/如何利用乳糖E.coli:“Ineedlactosepermease,β-galactosidase,andtransacetylase.”Whentouselactose/何时利用乳糖E.coli:

“Iwilluselactoseonlywhenthereisnoglucoseandthereislactose.”Glucose+Lactose+Glucose–Lactose–

Glucose–

Lactose+RegulatedExpressionof

LactoseMetabolizingGenes

乳糖代谢基因表达调控1.NegativeRegulation–thelacRepressor

负调控──lac阻遏蛋白2.PositiveRegulation–CAP

正调控──CAP2.NegativeRegulation–thelacRepressor负调控──lac阻遏蛋白NegativeRegulation–thelacRepressor

负调控──lac阻遏蛋白3.PositiveRegulation–CAP

正调控──CAPCAP-cAMPcomplexcAMPCAPDNACAPcAMPAdenylyl

Cyclase(AC)OO—CH2AOP——OOOHcAMPATPACGlucoseinhibitstheactivityofAC.CAP–“Theaccelerator”

lacrepressor–“Thebrake”Brake刹车Accelerator加速器Nowshowing

ThelacoperonFile:biophoto7\Genetics:AConceptualApproach\Thelacoperon(15’)3.3.3TheTrpOperon

/色氨酸操纵子NHCHCCH2CH+H3NCOO–NHCHCHCH3CH+H3NCOO–TryptophanOHOH1.NegativeRegulation–HighTrplevelHightryptophanlevel:NotranscriptionNegativeRegulation–lowTrplevelLowtryptophanlevel:Transcriptionoccurslacrepressorvs.

trprepressorPromoterLeader-attenuatortrpEtrpDAporepressordimerTryptophanRepressordimerRNApolymerasecannotbindtothepromoterOperator(b)Hightryptophan:transcriptionisrepressed.HightryptophanlevelLowlactoselevelPromoterLeader-attenuatortrpEtrpDAporepressordimerTryptophanRepressordimerRNApolymerasecannotbindtothepromoterOperator(b)Hightryptophan:transcriptionisrepressed.Lactose–theinducer

Tryptophan–theco-repressorInducer诱导物Co-repressor辅阻遏物Nowshowing

ThetrpoperonFile:biophoto7\Life\Thetrpoperon(6’)TheTrpOperon:Nopositiveregulation2.Attenuation/衰减作用Notalltranscriptionisrepressed.Attenuator:“STOP!”Theleader-attenuatorregion

前导子-衰减子区域Theleader-attenuatorRNAThecoupledtranscription-translation偶联转录-翻译作用Whentryptophanlevelislow…Whentryptophanlevelishigh…Nowshowing

AttenuationFile:biophoto7\Genetics:AConceptualApproach\Attenuation(10’)3.3.4AraandGalOperons

3.3.4

阿拉伯糖操纵子与半乳糖操纵子半乳糖CHOHOCHHCOHHCOHCH2OHArabinose阿拉伯糖1.AraOperon

/阿拉伯糖操纵子Whenthereisnoarabinose,theoperonisrepressed.AraOperon

/阿拉伯糖操纵子Whenthereisarabinose,theoperonisinduced.2.GalOperon

/半乳糖操纵子Whenthereisnogalactose,theoperonisrepressed.GalOperon

/半乳糖操纵子Whenthereisgalactose,theoperonisinduced.3.4Experiments/实验研究ThelacoperonConstitutivemutantlacI–

/组成型突变体lacI–Thelostfunctionwascompensated.ConstitutivemutantlacOC

/组成型突变体lacOCThelostfunctionwasNOTcompensated.LacOcfunctionsonlyonthesameDNAfragmentStructureofthelacoperon

乳糖操纵子的结构Chapter4TranscriptioninEukaryotes:MechanismandRegulation4.1EukaryoticRNApolymerases4.2EukaryoticPromoters4.3GeneralTranscriptionFactorsandInitiation4.4SpecificTranscriptionFactorsandTranscriptionalRegulation4.5Structuresofspecifictranscriptionfactors4.6Experiments4.1真核生物RNA聚合酶4.2真核生物启动子4.3通用转录因子与转录起始4.4特异转录因子与转录调控4.5特异转录因子的结构4.6实验研究 TranscriptioninEukaryotes:MechanismandRegulation

真核生物转录：机理与调控ProkaryoteEukaryoteTranscriptioninprokaryotesTranscriptionineukaryotesEukaryoticRNApolymeraseIIandtranscriptionfactorsSpecifictranscriptionfactorGeneraltranscriptionfactorsGeneraltranscriptionfactors4.1EukaryoticRNApolymerases

真核生物RNA聚合酶LocationsofRNApolymerasesNucleolus(RNApolymeraseI)Nucleoplasm(RNApolymeraseIIandIII)RNApolymeraseIRNApolymeraseIrRNAprecursorProcessing5.8SrRNA18SrRNA28SrRNARNApolymeraseIIIRNApolymeraseIIIRNAprecursorProcessing5SrRNA4.5StRNAU6snRNARNApolymeraseIIRNApolymeraseIIRNAprecursorProcessingsnRNAsTranslationProteinmRNARNApolymeraseIIHoloenzyme:

Coreenzyme(β’,β&α)+SubunitσRNApolII:

Coresubunits(1,2&3)+Subunits4-12RNApolymeraseII4.2EukaryoticPromoters

真核生物启动子ProkaryoticpromoterEukaryoticpromoterEukaryoticPromoters

真核生物启动子“IwouldliketohaveTATAboxandInitiatorasmypromoter.Whatwouldyoulike?”“Well,IpreferBREandDPE.”TATAboxTATAAA-10boxTATAATSpecializedgeneshaveTATAboxesTATAboxSpecializedgenes+Housekeepinggenes–Genesofdevelopment–DPE:DownstreamPromoterElementGCGATInitiator/起始子PyPyANPyPyTABRE:TFIIB

RecognitionElementCGCCGGGCCA4.3GeneralTranscriptionFactorsand

Initiation（通用转录因子与转录起始）ProkaryoticRNApolymerasepre-initiationcomplexEukaryoticRNApolymeraseII:“Ineedhelpfromotherproteins.”4.3.1TFIIDTBP:TATA-BindingProteinShow3DstructureofTBPFile:TBP-TATAboxcomplex1.valTAFIIs:TBP-AssociatedFactors

IITAFIIs:1)BindingtoDPEorInrTAFIIs:2)BindingtoActivator4.3.2OtherTFIIs

Nowshowing

InitiationofTranscriptionFile:biophoto7\Life\Initiationoftranscription(5’)4.3.3GeneralTranscriptionfactors

forRNAPolymeraseIandIII

RNA聚合酶I和III的通用转录因子1.GeneralTFsofRNApolymeraseI2.GeneralTFsofRNApolymeraseIIIGeneralTFsofRNApolymeraseIII4.4SpecificTranscriptionFactorsandTranscriptionalRegulation

特异转录因子与转录调控SpecifictranscriptionfactorSpecificTranscriptionFactorsandTranscriptionalRegulation

特异转录因子与转录调控4.4.1Activators/激活蛋白4.4.2Repressors/阻遏蛋白4.4.1Activators/激活蛋白Specifictranscriptionfactorsthatenhancetherateoftranscriptionarecalledactivators.能提高转录速率的特异转录因子称为激活蛋白。Helprecruitthepre-initiationcomplex.Helporganizethesubunitsofthepre-initiationcomplex,andmaintaincohesionofthecomplex.AttractenzymestoloosenDNAfromproteins,freeingimportantregionslikethepromoter.1.Helprecruitthepre-initiationcomplex2.Helporganizethesubunitsofthepre-initiationcomplex3.AttractenzymestoloosenDNA4.4.2Repressors/阻遏蛋白Specifictranscriptionfactorsthatpreventthetranscriptionofagenearecalledrepressors.能阻碍基因转录的特异转录因子称为阻遏蛋白。Blockingtheactivityofactivators.Blockingaccessofthepre-initiationcomplextothepromoter.RecruitingenzymesthattightenDNAaroundproteins.1.Blockingtheactivityofactivators2.Blockingaccessofthepre-initiationcomplextothepromoter.3.RecruitingenzymesthattightenDNAaroundproteins4.4.3EnhancersandSilencers

增强子和沉默子SpecifictranscriptionfactorsrelyonDNAsequencestotellthemhoweachgeneshouldberegulated.Thesesequencesarecalledenhancersorsilencers,dependingwhethertheybindactivatorsorrepressors,respectively.特异转录因子依靠DNA序列来告诉它们应该怎样调控每一种基因。这些序列称为增强子或沉默子，分别依赖于它们结合的是激活蛋白还是阻遏蛋白。EnhancersandSilencersTranscriptionisactivatedEnhancerTranscriptionisrepressedSilencerRepressorActivatorDNAlooping/DNA成环SeaUrchinEndo16geneAredseaurchin红海胆4.5StructuresofSpecificTranscriptionFactors

4.5特异转录因子的结构??ProkaryoticEukaryoticMajorGroovevs.MinorGrooveMajorgrooveA≠TG≠CMinorgrooveA≈TG≈C4.5.1DNA-BindingMotifsinProkaryotes

原核生物DNA结合基序RecognitionhelixThehelix-turn-helix(HTH)motifNegativeRegulation–thelacRepressor

负调控──lac阻遏蛋白Show3DstructureofthelacrepressorFile:lacrepressorwithDNA.valThetrprepressorThetrprepressorTrpTrprepressorDNAE.coliOperatorShow3DstructureofthetrprepressorFile:trprepressor.val4.5.2DNA-BindingMotifsineukaryote 真核生物DNA结合基序1.Thehomeodomain

同源异型域2.Zincfingermotif

锌指基序3.Leucinezipper

亮氨酸拉链HLHmotif

螺旋-环-螺旋基序?Eukaryotic1.Thehomeodomain

/同源异型域SimilartoHTHofprokaryotesMutationofhomeoticgenesHomeoticgenesShow3DstructureofhomeodomainFile:Homeodomain.valBaird-TitusJM,etal.

ThesolutionstructureofthenativeK50BicoidhomeodomainboundtotheconsensusTAATCCDNA-bindingsite

J.Mol.Biol.v356,p.1137-1151

2.Zincfingermotif/锌指基序Show3DstructureofzincfingersFinger1Finger2Finger3File:Zincfinger.val3.Leucinezipper/亮氨酸拉链LeucinesHeterodimerization

/异源二聚化作用Show3DstructureofleucinezipperWuSW,etal.DesignandcharacterizationofamultimericDNAbindingproteinusingSac7dandGCN4astemplates

Proteinsv60,p.617-628

File:Leucinezipper.valYeastcellswww.zeiss.de/C12567BE0045ACF1/ContentsWWWInte

4.Helix-loop-helixmotif/螺旋-