Chapter3/第3章Chapter3 TranscriptioninProkaryotes:MechanismandRegulation3.1WhyUseanRNAIntermediate?3.2MechanismofTranscription3.3RegulationofGeneExpressioninProkaryotes3.4Experiments第3章原核生物轉(zhuǎn)錄：機(jī)理與調(diào)控3.1為什么使用RNA

作為中間物？3.2轉(zhuǎn)錄機(jī)理3.3原核生物基因表達(dá)調(diào)控3.4實(shí)驗(yàn)研究3.1WhyUseanRNAintermediate?

3.1為什么使用RNA作為中間物？ChromosomesarelargeandcomplexLowefficiency!DNAProteinRibosomeDNAmustbestableeOnemRNAmanyproteinsMoreopportunitiesforregulationChapter3Chapter4DiscussionIsthereanydisadvantageswhenRNAisusedastheintermediate?3.2MechanismofTranscriptioninprokaryotes

3.2轉(zhuǎn)錄機(jī)理Questions1.Wheredoestranscriptionbegin?2.Wheredoestranscriptionend?3.Whichstrandofthedouble-strandDNAistranscriptedandwhy?4.Areprimersneededintranscription?SubstancesusedintranscriptioninprokaryotesMaterials:NTP(ATP,UTP,GTP,CTP)Template:DNAEnzyme:RNApolymerase,RNA-polOtherproteinfactors3.2.1Promoters /啟動(dòng)子ThepromotersDNAsequencesorsitesupstreamofgenes,whichisrecognizedandboundbytheRNApolymerasetoinitiatethetranscription,isknownaspromoter.Thesequencesofpromotersareoftenconservedbetweendifferentgenes,whichgiverisetodifferentefficienciesofgeneexpression.?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(shí)際的序列與它相差很小或完全相同。）1.DoallofthepromotersofdifferentgeneshavethesamesequenceinE.coli?

Discussion3.2.2RNAPolymerase/RNA聚合酶Coreenzymevs.HoloenzymeHoloenzymestartstranscriptionatpromotersCoreenzymestartstranscriptionatrandom1.DoesthecoreenzymeofRNApolymerasealwaysbindwithσfactorinE.coli?

DiscussionquestionsIfnot,doesthetranscriptionhappenatrandomsitesinE.colijustasinvitro?

2.CantheσfactorbindtothepromotersintheabsenceofcoreenzymeofRNApolymerase?

3.WhatrolesdotheotherthreesubunitspossessinRNApolymerase?CatalyticareaofRNApolymeraseRNApolymeraseresemblesacrabclaw3.2.3TranscriptionMechanism

3.2.3轉(zhuǎn)錄機(jī)理Initiation/起始

Elongation/延伸

Termination/終止1.Initiation/起始Step1Step2Step3Step4Canyouexplainwhythefirstsynthesizedshorttranscriptsaregivenup?

Discussionquestion2.Elongation/延伸TranscriptionbubbleWouldyoupleasedescribethetranscriptionbubbleindetail?

DiscussionquestionThepolymerizationreaction

聚合反應(yīng)RNAsynthesis:5’→3’Proofreading/校正UCRemoveUAddCHowdoyouexplaintheproofreadingabilityofRNA?

DiscussionquestionSomeerrorsaresometimesintroducedintotheRNAeventhoughRNApolymerasehasproofreadingability.AretheseincorporatederrorsvitaltoE.coliandwhy?

Discussionquestion1.RNAlifetimeisnotlong

2.Proteinsaredegradedquickly

3.Somemutationshavelittleeffectonthefunctionofaprotein.3.Termination/終止Intrinsictermination內(nèi)在型終止ρ-dependentterminationρ依賴型終止Intrinsictermination/內(nèi)在型終止

Basepair/堿基對(duì)ρ-dependenttermination/ρ依賴型終止

RhoproteinsX-RayCrystalStructureOfRhoprotein[MMDBID:24381]

Anewtranscriptionbeginsbeforetheformertranscriptionends53DNA原核生物轉(zhuǎn)錄過程中的羽毛狀現(xiàn)象RibosomeRNARNApolymerase在同一DNA模板上，有多個(gè)轉(zhuǎn)錄同時(shí)在進(jìn)行；轉(zhuǎn)錄尚未完成，翻譯已在進(jìn)行。這種形狀說明：Nowshowing

BacterialTranscriptionFile:biophoto7\Genetics:AConceptualApproach\Bacterialtranscription(6’).AstalledRNApolymerasecanbereleasedbycleavingthe3`endofthetranscript.RNApolymerasecanrecoverfrompausingRNApolymeraseduringelongationRNApolymeraseisstalled&backtracks3`regionofRNAiscleavedNew3`endislocatedincatalyticsiteCatalyticsiteresumeselongationNowshowing

TranscriptionFile:biophoto7\Life\Transcription.(5’)3.3Regulationofgeneexpression

inprokaryotes

3.3原核生物基因表達(dá)調(diào)控3.3.1CoordinateRegulation3.3.2TheLacOperon3.3.3TheTrpOperon3.3.4Ara&GalOperons3.3.1協(xié)同調(diào)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(dá)調(diào)控ConditionARegulationofgeneexpression

基因表達(dá)調(diào)控ConditionB3.3.1CoordinateRegulation

協(xié)同調(diào)控Operator操縱基因Whatiscoordinateregulation?Inprokaryotes,genesthatsharearelatedfunctionareoftenlocatedadjacenttoeachotherandareturnedonorofftogether.Wesaythatthesegenesareundercoordinateregulation.PolycistronicmRNA

多順反子mRNAPolycistronicmRNATranslationProtein1Protein2Protein3WhatispolycistronicmRNA?

ProkaryoticRNApolymerasetranscribesallofthegenesinanoperonintoonelongmRNA.ThismRNAiscalledpolycistronicmRNAbecauseitcodesformultipleproteins.Cistronisanotherwordforgenes.Betweeneachcistronthereareshortuntranslatedregionsthatcontainsignalstostopandstarttranslation.Thisallowsthegenestobetranslatedasseparateproteins,insteadofonelongprotein.3.3.2ThelacOperon/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(shí)利用乳糖E.coli:

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

Glucose–

Lactose+RegulatedExpressionof

LactoseMetabolizingGenes

乳糖代謝基因表達(dá)調(diào)控1.NegativeRegulation–thelacRepressor

負(fù)調(diào)控──lac阻遏蛋白2.PositiveRegulation–CAP

正調(diào)控──CAP2.NegativeRegulation–thelacRepressor負(fù)調(diào)控──lac阻遏蛋白NegativeRegulation–thelacRepressor

負(fù)調(diào)控──lac阻遏蛋白3.PositiveRegulation–CAP

正調(diào)控──CAPCAP-cAMPcomplexcAMPCAPDNACAPcAMPAdenylylCyclase(AC)OO—CH2AOP——OOOHcAMPATPACGlucoseinhibitstheactivityofAC.CAP–“Theaccelerator”

lacrepressor–“Thebrake”Brake剎車Accelerator加速器NegativeregulationandpositiveregulationIftranscriptionispreventedafteraregulatoryproteinbindstotheregulatoryregioninagene,Thiskindofregulationiscallednegativeregulation.Otherwiseitiscalledpositiveregulation.Questions1.IsthereanyGlucoseoperonandwhy?2.Whydothebacteriauseglucosebutnotlactosewhenbothoftheglucoseandlactoseexistareavailable?3.Istheexpressionoflacrepressorregulatedbylactose?4.Doesthereexistanylactosepermease,β-galactosidaseinthecellwhenthereisnolactose?Whatisoperon?Inprokaryotes,thefunctionally-relatedgenesarelocatedadjacenttoeachotherontheDNA.Theyarealsoregulatedtogetherunderthecontrolofoneregulatoryregion.Thisregulatoryregioniscomposedofonepromoterandoperator.Thisunitofprokaryoticgeneexpressioniscalledoperon.Whatisoperator?OperatorisacontrolDNAsequenceupstreamofthestructuralgenesthatregulatesthetranscriptionofthestructuralgenes.Whatiscalledinducedexpression?

Figure8UsingaPBAD.Theexpressionofthetargetproteinwasinducedwithincreasingconcentrationsofarabinose.Example:theinducedexpressionanditsimportanceinthegeneengineeringCulturetime(h)Concentrationofbacteria(mg/ml)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(dǎo)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(dǎo)子-衰減子區(qū)域Theleader-attenuatorRNAUGGUGGUGGUGGUGGUGGUAGUGAUGAThecoupledtranscription-translation偶聯(lián)轉(zhuǎn)錄-翻譯作用Whentryptophanlevelislow…UGAWhentryptophanlevelishigh…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CHOHOCHHCOHH