Chapter3/第3章Chapter3 TranscriptioninProkaryotes:MechanismandRegulation3.1WhyUseanRNAIntermediate?3.2MechanismofTranscription3.3RegulationofGeneExpressioninProkaryotes3.4Experiments第3章原核生物轉錄：機理與調控3.1為什么使用RNA

作為中間物？3.2轉錄機理3.3原核生物基因表達調控3.4實驗研究3.1WhyUseanRNAintermediate?

3.1為什么使用RNA作為中間物？ChromosomesarelargeandcomplexLowefficiency!DNAProteinRibosomeDNAmustbestableeOnemRNAmanyproteinsMoreopportunitiesforregulationChapter3Chapter43.2MechanismofTranscription

3.2轉錄機理3.2.1Promoters /啟動子?10boxand?35boxCCAGGCTTTACACTT---CGTATGTTGTGTGGAATTCTTTTTGATGCAATT---ACTATAATAGACAGGGTAGGCGGTGTTGACATA---GTGATACTGAGCACATCA

-35box-10box

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

-35box-10box

+1TATAATTTGACAConsensussequencesConsensussequence:ageneralizedsequencefromwhichmostactualsequencesdifferverylittleornotatall.（共有序列：一種普遍的序列，大多數(shù)實際的序列與它相差很小或完全相同。）3.2.2RNAPolymerase/RNA聚合酶Coreenzymevs.HoloenzymeHoloenzymestartstranscriptionatpromotersCoreenzymestartstranscriptionatrandomCatalyticareaofRNApolymerase3.2.3TranscriptionMechanism

3.2.3轉錄機理Initiation/起始

Elongation/延伸

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

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

TranscriptionFile:biophoto7\Life\Transcription.(5’)3.Termination/終止Intrinsictermination內在型終止ρ-dependentterminationρ依賴型終止Intrinsictermination/內在型終止

Basepair/堿基對ρ-dependenttermination/ρ依賴型終止

RhoproteinsX-RayCrystalStructureOfRhoprotein[MMDBID:24381]

Nowshowing

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

inprokaryotes

3.3原核生物基因表達調控3.3.1CoordinateRegulation3.3.2TheLacOperon3.3.3TheTrpOperon3.3.4Ara&GalOperons3.3.1協(xié)同調控3.3.2乳糖操縱子3.3.3色氨酸操縱子3.3.4阿拉伯糖與半乳糖操縱子Regulationofgeneexpression

基因表達調控ConditionARegulationofgeneexpression

基因表達調控ConditionB3.3.1CoordinateRegulation

協(xié)同調控Operator操縱基因PolycistronicmRNA

多順反子mRNAPolycistronicmRNATranslationProtein1Protein2Protein33.3.2ThelacOperon

/乳糖操縱子Thelacoperon1.TheConditionsoflactoseMetabolism

1.

乳糖代謝的條件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/何時利用乳糖E.coli:

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

Glucose–

Lactose+RegulatedExpressionof

LactoseMetabolizingGenes

乳糖代謝基因表達調控1.NegativeRegulation–thelacRepressor

負調控──lac阻遏蛋白2.PositiveRegulation–CAP

正調控──CAP2.NegativeRegulation–thelacRepressor負調控──lac阻遏蛋白NegativeRegulation–thelacRepressor

負調控──lac阻遏蛋白3.PositiveRegulation–CAP

正調控──CAPCAP-cAMPcomplexcAMPCAPDNACAPcAMPAdenylyl

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

lacrepressor–“Thebrake”Brake剎車Accelerator加速器Nowshowing

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

/色氨酸操縱子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誘導物Co-repressor輔阻遏物Nowshowing

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

前導子-衰減子區(qū)域Theleader-attenuatorRNAThecoupledtranscription-translation偶聯(lián)轉錄-翻譯作用Whentryptophanlevelislow…Whentryptophanlevelishigh…Nowshowing

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

3.3.4

阿拉伯糖操縱子與半乳糖操縱子半乳糖CHOHOCHHCOHHCOHCH2OHArabinose阿拉伯糖1.AraOperon

/阿拉伯糖操縱子Whenthereisnoarabinose,theoperonisrepressed.AraOperon

/阿拉伯糖操縱子Whenthereisarabinose,theoperonisinduced.2.GalOperon

/半乳糖操縱子Whenthereisnogalactose,theoperonisrepressed.GalOperon

/半乳糖操縱子Whenthereisgalactose,theoperonisinduced.3.4Experiments/實驗研究ThelacoperonConstitutivemutantlacI–

/組成型突變體lacI–Thelostfunctionwascompensated.ConstitutivemutantlacOC

/組成型突變體lacOCThelostfunctionwasNOTcompensated.LacOcfunctionsonlyonthesameDNAfragmentStructureofthelacoperon

乳糖操縱子的結構Chapter4TranscriptioninEukaryotes:MechanismandRegulation4.1EukaryoticRNApolymerases4.2EukaryoticPromoters4.3GeneralTranscriptionFactorsandInitiation4.4SpecificTranscriptionFactorsandTranscriptionalRegulation4.5Structuresofspecifictranscriptionfactors4.6Experiments4.1真核生物RNA聚合酶4.2真核生物啟動子4.3通用轉錄因子與轉錄起始4.4特異轉錄因子與轉錄調控4.5特異轉錄因子的結構4.6實驗研究 TranscriptioninEukaryotes:MechanismandRegulation

真核生物轉錄：機理與調控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

真核生物啟動子ProkaryoticpromoterEukaryoticpromoterEukaryoticPromoters

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

RecognitionElementCGCCGGGCCA4.3GeneralTranscriptionFactorsand

Initiation（通用轉錄因子與轉錄起始）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的通用轉錄因子1.GeneralTFsofRNApolymeraseI2.GeneralTFsofRNApolymeraseIIIGeneralTFsofRNApolymeraseIII4.4SpecificTranscriptionFactorsandTranscriptionalRegulation

特異轉錄因子與轉錄調控SpecifictranscriptionfactorSpecificTranscriptionFactorsandTranscriptionalRegulation

特異轉錄因子與轉錄調控4.4.1Activators/激活蛋白4.4.2Repressors/阻遏蛋白4.4.1Activators/激活蛋白Specifictranscriptionfactorsthatenhancetherateoftranscriptionarecalledactivators.能提高轉錄速率的特異轉錄因子稱為激活蛋白。Helprecruitthepre-initiationcomplex.Helporganizethesubunitsofthepre-initiationcomplex,andmaintaincohesionofthecomplex.AttractenzymestoloosenDNAfromproteins,freeingimportantregionslikethepromoter.1.Helprecruitthepre-initiationcomplex2.Helporganizethesubunitsofthepre-initiationcomplex3.AttractenzymestoloosenDNA4.4.2Repressors/阻遏蛋白Specifictranscriptionfactorsthatpreventthetranscriptionofagenearecalledrepressors.能阻礙基因轉錄的特異轉錄因子稱為阻遏蛋白。Blockingtheactivityofactivators.Blockingaccessofthepre-initiationcomplextothepromoter.RecruitingenzymesthattightenDNAaroundproteins.1.Blockingtheactivityofactivators2.Blockingaccessofthepre-initiationcomplextothepromoter.3.RecruitingenzymesthattightenDNAaroundproteins4.4.3EnhancersandSilencers

增強子和沉默子SpecifictranscriptionfactorsrelyonDNAsequencestotellthemhoweachgeneshouldberegulated.Thesesequencesarecalledenhancersorsilencers,dependingwhethertheybindactivatorsorrepressors,respectively.特異轉錄因子依靠DNA序列來告訴它們應該怎樣調控每一種基因。這些序列稱為增強子或沉默子，分別依賴于它們結合的是激活蛋白還是阻遏蛋白。EnhancersandSilencersTranscriptionisactivatedEnhancerTranscriptionisrepressedSilencerRepressorActivatorDNAlooping/DNA成環(huán)SeaUrchinEndo16geneAredseaurchin紅海膽4.5StructuresofSpecificTranscriptionFactors

4.5特異轉錄因子的結構??ProkaryoticEukaryoticMajorGroovevs.MinorGrooveMajorgrooveA≠TG≠CMinorgrooveA≈TG≈C4.5.1DNA-BindingMotifsinProkaryotes

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

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

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?Eukaryotic1.Thehomeodomain

/同源異型域SimilartoHTHofprokaryotesMutationofhomeoticgenesHomeoticgenesShow3DstructureofhomeodomainFile:Homeodomain.valBaird-TitusJM,etal.

ThesolutionstructureofthenativeK50BicoidhomeodomainboundtotheconsensusTAATCCDNA-bindingsite

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

2.Zincfingermotif/鋅指基序Show3DstructureofzincfingersFinger1Finger2Finger3File:Zincfinger.val3.Leucinezipper/亮氨酸拉鏈LeucinesHeterodimerization

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

Proteinsv60,p.617-628

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

4.Helix-loop-helixmotif/螺旋-