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第十章GeneRegulationinProkaryotesTOPIC1

PrinciplesofTranscriptionalRegulationTOPIC2RegulationofTranscriptionInitiation:ExamplesfromBacteria(Lacoperon,乳糖操纵子)TOPIC3ExamplesofGeneRegulationafterTranscriptionInitiation(Trpoperon,色氨酸操纵子)Topic1:Principles

ofTranscriptionRegulation1.Geneexpressioniscontrolledatdifferentstages

(基因表达调控可以发生在不同时期)ThekeystepofgeneregulationtakesplaceattheinitiationoftranscriptionGeneregulationcanhappenatdifferentlevels:TranscriptionalregulationPost-TranscriptionalregulationTranslationalregulationTranscriptioninitiationBinding(closedcomplex)Promoter“melting”(opencomplex)InitialtranscriptionKeystepofregulation2.GeneExpressionisControlledbyRegulatoryProteins(调控蛋白)GeneexpressionisveryoftencontrolledbyExtracellularSignals,

whicharecommunicatedtogenesbyregulatoryproteins:PositiveregulatorsoractivatorsINCREASEthetranscriptionNegativeregulatorsorrepressors

DECREASEorELIMINATEthetranscription3.Manypromotersareregulatedbyactivators

(激活蛋白)

thathelpRNAPbindDNA(recruitment)andbyrepressors

(阻遏蛋白)

thatblockthebinding.RNAPbindsmanypromotersweakly,activatorsthatcontaintwobindingsitestobindaDNAsequenceandRNAPsimultaneouslycanenhancetheRNAPaffinitywiththepromoters,andthusincreasesgenetranscription.Thisiscalledrecruitmentregulation(招募调控).

Onthecontrary,Repressorscanbindtotheoperatorinsideofthepromoterregion,whichpreventsRNAPbindingandthetranscriptionofthetargetgene.a.AbsenceofRegulatoryProteins:basallevelexpressionb.Repressorbindingtotheoperatorrepressesexpressionc.ActivatorbindingactivatesexpressionFig16-14

Allosteryregulation(异构调控)

aftertheRNAPolymeraseBinding Insomecases,RNAPbindsthepromotersefficiently,butnospontaneousisomerizationoccurstoleadtotheopencomplex,resultinginnoorlowtranscription.

Someactivatorscanbindtotheclosedcomplex,inducingconformationalchangeineitherRNAPorDNApromoter,whichconvertstheclosedcomplextoopencomplexandthuspromotesthetranscription.AllosteryregulationControlelementStructuralgenesBasicprincipleofregulationofgeneexpression:Interactionbetweentrans-actingfactor

(regulatoryprotein)andcis-actingelement

(controlelement)反式作用因子和顺式作用元件之间的相互作用trans-actingfactorTopic2:RegulationofTranscriptionInitiation:Examples

fromBacteriaFirstexample:LacoperonThelactoseOperon(乳糖操纵子)FrançoisJacob

JacquesMonod

FrenchscientistsJacobandMonod(1965NobelPrize)Operon(操纵子,操纵元):

aunitofprokarytoicgeneexpressionandregulationwhichtypicallyincludes:1.

Structuralgenes

forenzymesinaspecificbiosyntheticpathwaywhoseexpressioniscoordinatelycontrolled

2.

Controlelements,suchasoperator(操作子)sequence-cis-actingelement

3.

Regulatorgene(s)

codeforregulatoryprotein

whichcanrecognizeandinteractwiththecontrolelementsOutlineEstablishmentofLacoperonNegativecontroloftheLacoperon:interactionbetweenrepressorandoperator(负调控)PositivecontroloftheLacoperon:interactionbetweenCAP-cAMPcomplexandCAPbindingsite(正调控)EstablishmentofLacoperon

fromphenomenontomechanism现象:乳糖代谢的可诱导性Inductionofenzyme(酶的诱导现象)Addlactoseremovelactosetime/minβ-galactosidase(半乳糖苷酶)galactosidepermease(半乳糖苷渗透酶)β-galactosidaseactivityappearedonlywhenlactosewasprovidedinthegrowthmediumβ-galactosidasegene代谢乳糖的酶能够被乳糖所诱导激活lacYencodesacellmembraneproteincalledgalactosidepermease(半乳糖苷渗透酶)totransportLactoseacrossthecellwalllacZcodesforβ-galactosidase(半乳糖苷酶)forlactosehydrolysislacAencodesathiogalactosidetransacetylase(硫代半乳糖苷转乙酰酶)togetridofthetoxicthiogalacosidesTheLACoperonStructuralgeneclusters(functionalrelated)arecoordinatelycontrolled(协同调控)polycistronicmRNA(多顺反子)EconomicandefficientprinciplequestionWhatisthemechanismofinductionofenzyme?HowwasthelacZ,lacYandlacAgeneregulatedatthetranscriptionlevel?FrenchscientistsJacobandMonoddissectedthisproblemgeneticallyandputforwardtheoperonmodel(1965NobelPrize)PerfectcombinationofgeneticandbiochemicalmethodIsolatedmutantsofE.colithatmadeβ-galactosidaseirrespectiveofwhetherlactosewaspresent(诱导型(Mutantsinwhichtheenzymewasproducedconstitutively)ResearchstrategyofJacobandMonod

Twoclassmutants(组成型突变):两种突变位点I.thegeneencodingtheLacrepressor(LacI)wasinactivatedII.theoperatorsite(LacO)wasmutatedQuestion:whatisthemechanismofLacIandLacOregulatingtheexpressionofLacZYA?组成型)Twokeyelements:

lacI:encodinglacrepressor,trans-actingfactoroperator(lacO):lacrepressorbindingsite,cis-actingelementBasicmodelofgenetranscriptionregulation:trans-regulatoryfactorsinteractwithcis-actingelementOperon:操纵子,操纵元Operator:操作子ControlelementStructuralgenesBasicprincipleofregulationofgeneexpression:Interactionbetweentrans-actingfactor

(regulatoryprotein)andcis-actingelement

(controlelement)反式作用因子和顺式作用元件之间的相互作用trans-actingfactor二NegativecontroloftheLacoperon:interactionbetweenrepressorandoperator(负调控)

lacIpromoterRNApolymeraselacZlacYlacANotranscriptiontranscriptionoperatoractiverepressorinactiverepressor阻遏去阻遏Inducer:

isasmallmoleculethattriggersgenetranscriptionbybindingtorepressor-去阻遏作用TherealinducerinLacoperonisallolactose(异构乳糖)Lactoseisconvertedtoallolactosebyβ-galactosidase,thereforelactosecanindirectlyturnofftherepressorWhatistheinducer?allolactose(异构乳糖)Widelyusedinducer:1PurificationofLacrepressorStrategy:InducerbindingtorepressorAffinitychromatography(亲和层析)canfacilitatemorerapidproteinpurification thetargetproteinisknowntoestablishaspecificandhigh-affinityinteractionwithaspecificprotein/nucleicacids/smallmoleculewecancouplethisspecificpartnerofthetargetproteintothecolumnandthusthetargetproteinwillbeselectivelyboundtothecolumn.Thismethodiscalledaffinitychromatography.Lacrepressor结合2Testwhethertherepressorcaninteractwithoperator(lacO)

Filterbinding(滤膜结合法)StudytheinteractionbetweenproteinandDNAWidelyusedinducer:O+OCλphageDNABiochemicalexperimentPerfectcombinationofgeneticandbiochemicalmethodGelretardation(凝胶阻滞,EMSA)ConfirmationofthespecificinteractionbetweenisolatedproteinandregulatoryelementElectrophoreticMobilityShiftAssay,EMSA(电泳迁移率变动分析)

DNAboundtotwoproteinsDNA-proteincomplexBareDNAADNAboundwithmorethanoneproteintoformalargercomplexBareDNADNA-proteincomplexgelelectrophoresis放射自显影:检测DNA-protein复合物标记特异DNA未标记特异DNAprotein随机DNA序列菠菜叶绿体蛋白质提取物与高粱psbA启动子DNA的竞争结合实验:

A~E——未标记的竞争DNA量分别是标记DNA探针量的1,5,10,30,50倍。

竞争性EMSApromoteroperator(反向对称序列)OperatoroverlapspromoterThesiteboundbylacrepressoriscalledthelacoperator(Olac

),andtheOlac

overlapspromoter(Plac)ThereforerepressorboundtotheoperatorphysicallypreventsRNApolymerasefrombindingtothepromoterLacrepressorboundtotheoperatorpreventsRNAPfrombindingtothepromoterrepressorOperatormutations(Oc)areconstitutivebecausetheoperatorisunabletobindrepressorprotein;thisallowsRNApolymerase

tohaveunrestrainedaccesstothepromoterRNApolymeraselacImRNAproteinquestionWhatistheeffectofspecificbasemutationinoperatorontheinteraction

betweenoperatorandrepressor?Whichmethodcanyouusetoresolveabovequestion?promoteroperator(反向对称序列)Operatoroverlapspromoter用不同突变碱基的标记探针DNA做EMSA分析评估特异性碱基突变对探针DNA(operator)和结合蛋白(repressor)相互作用(亲和力)的影响Gelretardation(凝胶阻滞,EMSA)BareDNADNA-proteincomplexgelelectrophoresis放射自显影:检测DNA-protein复合物标记特异DNA未标记特异DNAprotein随机DNA序列SummaryNegativecontroloftheLacoperon:interactionbetweenrepressorandoperator(阻遏作用)Inducer:allolactosebindingtorepressorresultedintheinactivationofrepressor(去阻遏作用)wideapplicationingeneengineering(IPTGinducingexpression)三PositivecontroloftheLacoperon:interactionbetweenCAP-cAMPcomplexandCAPbindingsite(正调控)

fromphenomenontomechanism面包窝窝头I.FromonephenomenontothecontentofthisclassI.Fromonephenomenontothecontentofthisclass大肠杆菌选择食物的优先顺序Why?PositivecontroloftheLacoperon(乳糖操纵子的正调控)先吃葡萄糖后吃乳糖正调控:油门,加速器Whatispositivecontrol?建立大肠杆菌乳糖操纵子正调控机制的研究历程与研究思路fromphenomenontomechanism大肠杆菌乳糖操纵子正调控机制的发现:Whengrowninthemediumcontainingbothglucoseandlactose,E.colicellswouldnotuselactoseuntilglucosewasexhausted.TheexpressionoflacoperonwasinhibitedbyglucoseglucoseusedlactoseusedGlucoseexhaustedLacoperonβ-半乳糖苷酶β-半乳糖苷酶活性Phenomenon:大肠杆菌的二次生长曲线(E.coli

diauxicgrowth)HowistheLacoperonstimulatedafterglucoseisexhausted?当葡萄糖耗尽时,乳糖操纵子的表达是如何被激活的?

KeyQuestion:glucosecAMPglucosecAMP葡萄糖浓度上升,cAMP浓度下降葡萄糖浓度下降,cAMP浓度上升cAMPisthesignalmolecularthatrespondstotheglucoseconcentration--信号分子(cAMP,环腺苷酸)cAMP在糖代谢过程中究竟发挥什么样的作用呢?NextQuestion:+glucose+lactose-cAMP(不加cAMP)ExpressionofLacoperoninE.coli

wasinhibitedbyglucose

10203040Time(min)050+glucose+lactose+cAMP(添加大量的cAMP)ExpressionofLacoperoninE.coli

wasstimulatedbycAMP10203040Time(min)050β-半乳糖苷酶活性始终为0β-半乳糖苷酶活性显著上升乳糖操纵子的表达能够被cAMP分子所激活ThisfindingimplicatedstronglythatcAMPwasinvolvedinthepositivecontrolofthelacoperonNextquestion:WhycancAMPactivatetheexpressionofLacoperon?WhatisthemechanismbehindthecAMPactingasthepositiveregulatoryfactor?

cAMP分子为什么能够激活乳糖操纵子的表达?也就是说cAMP分子作为正调控因子的作用机制是什么?如果你是正在研究乳糖代谢调控的研究者,下一步你会怎么做?ControlelementStructuralgenesBasicprincipleofregulationofgeneexpression:Interactionbetweentrans-actingfactor(regulatoryprotein)andcis-actingelement(controlelement)理论指导实践基因表达调控的基本机制FishingwithcAMPasthebait(钓鱼诱饵)bait

targetcAMPCAP(CataboliteActivatorProtein,代谢产物激活蛋白)Hypothesis:细胞内是否存在一种能够和cAMP结合的蛋白,进而通过这种蛋白来调控乳糖操纵子基因的表达呢?Experiment寻找能够和cAMP结合的蛋白Researchstrategy:lookingforthepositiveregulatoryprotein

involvedinpositivecontroloflacoperonbycAMPDiscoveryofCAP(CataboliteActivatorProtein,代谢产物激活蛋白)orCRP(cAMPReceptorProtein,cAMP受体蛋白)AffinitychromatographyCAP蛋白和cAMP的相互作用是否的确能够激活乳糖操纵子的表达?NextQuestion:Strategy:findingCAPmutantthatbindstocAMPmoreweaklythanwild-typeCAPGeneticmethod,mutationidea!乳糖操纵子的表达cAMP浓度野生型CAP蛋白突变型CAP蛋白结论:CAP蛋白和cAMP的相互作用的确能够激活乳糖操纵子的表达突变的思想CAPNotstimulatetheexpressionofLacoperonCAP-cAMPcomplexStimulatetheexpressionofLacoperoncAMPCAP-cAMPcomplex(CAP-cAMP复合体)CAP蛋白-cAMP复合体究竟是如何激活乳糖操纵子的表达?NextQuestion:ControlelementStructuralgenesRegulatorproteinCAP基因表达调控的基本机制DNA调控元件?相互作用HowwasthelacO(operator)siteidentified?IsolatedmutantsofE.colithatmadeβ-galactosidaseirrespectiveofwhetherlactosewaspresent(诱导型(Mutantsinwhichtheenzymewasproducedconstitutively)Twoclassmutants(组成型突变):I.thegeneencodingtheLacrepressor(LacI)wasinactivatedII.theoperatorsite(LacO)wasmutated组成型)Geneticmethod:lookingforaclassoflacmutantsinwhichCAP-cAMPcouldnotstimulatelactranscription—mutationideaThesemutationsmappedtotheCAP-cAMPbindingsite

locatingjustupstreamofthepromoterLookingforthecontrolelementinteractingwithCAP-cAMPCAPbindingsite(CAP蛋白结合位点)locatingjustupstreamofthepromoter(启动子)promoterCAPbindingsiteCAPbindingsitepromoterL1deletion:CAP-cAMPcouldnotstimulatelactranscriptionX-raystructureofCAP-cAMPboundtoCAPbindingsiteCAP-cAMP最新的结构生物学实验证据:CAPbindingsiteCAP-cAMPinteractwiththeCAP-cAMPbindingsite

stimulatelactranscriptionNextquestion:Whatisthemechanismbehindthisstimulation?Glucose

cAMPControlelementStructuralgenesBasicmechanismofregulationofgeneexpressionRegulatorproteinCAP-cAMPcomplexCAPbindingsiteCAP-cAMP复合体和CAPbindingsite之间的结合究竟是怎样激活乳糖操纵子的表达的?

CAP蛋白激活乳糖操纵子表达的分子作用机制究竟是什么?Lastquestion:TranscriptioninitiationBinding(closedcomplex)Promoter“melting”(opencomplex)InitialtranscriptionHypothesis:

CAP-cAMPcanstimulatetheformationofopenpromotercomplex?Howcanwetestthishypothesis?Rifampicin:利福平TranscriptioninitiationBinding(closedcomplex)Promoter“melting”(opencomplex)InitialtranscriptionClosedpromotercomplexSensitivetorifampicinOpenpromotercomplexresistanttorifampicinMechanismCAP-cAMPCAPbindingsitelacZTranscription(转录)促进了RNA聚合酶和启动子结合形成稳定的开放复合体(RNA聚合酶)(启动子)……CAP激活基因转录mRNAb-半乳糖苷酶b-半乳糖苷透性酶b-半乳糖苷乙酰基转移酶ActiveCAP-cAMPcomplexHighlevelofcAMP启动子结构基因lacZlacAlacYCAPbindingsiteRNApolymeraseglucoseabsent,lactosepresent(不存在葡萄糖,只存在乳糖)GlucoseUselactoseSummary:PositivecontroloftheLacoperonLacoperon转录翻译TheLACoperonPhenomenon:大肠杆菌二次生长曲线HowcanE.coliregulatetheorderofusingglucoseandlactose?cAMPwasinvolvedinthepositivecontroloftheLacoperonWhycancAMPactivatetheexpressionofLacoperon?questionquestion乳糖操纵子正调控研究思路总结:fromphenomenontomechanismDiscoveryofCAP(CataboliteActivatorProtein,代谢产物激活蛋白)orCRP(cAMPReceptorProtein,cAMP受体蛋白)TheCAP-cAMPcomplexisreallyimportanttopositivecontroloflacoperonbycAMPLookingforthecis-actingelementinteractingwithCAP-cAMPWhatisthemechanismbehindtheCAP-cAMPcomplexpositivelystimulatingthelacoperon?Discoveryof

CAP-cAMPbindingsiteWhatisthemechanismbehindthisstimulation?CAPactivatestheLactranscriptionthroughrecruitmentofRNAPtotheweakPlac促进开放启动子起始复合体的形成Basicprincipleofregulationofgeneexpression:Interactionbetweentrans-actingelement(regulatoryprotein)andcis-actingelement(controlelement)Mechanism:CAP-cAMPcanstimulatetheformationofopenpromotercomplex教学目的学习和掌握基本理论知识和基本概念领会科学研究的精髓,掌握科学研究的基本方法,培养学生提出问题、分析问题和解决问题的能力

提升科学素养,训练研究性的思维能力

科学研究方法和科学思维方式的学习ScientificresearchPhenomenonQuestionHypothesisExperimentMechanismSummary:ImportantbasicconceptsOperonOperatorNegativecontrol:repressorPositivecontrol:activatorRecruitmentregulationAllostericallyregulationTrans-actingfactorCis-actingelementTopic3:ExamplesofGeneRegulationatStepsAfterTranscriptionInitiationTranscriptioninitiationBinding(closedcomplex)Promoter“melting”(opencomplex)Initialtranscriptionexample:thetryptophanoperon(色氨酸操纵子)Aminoacidbiosyntheticmetabolism(合成代谢)分枝酸色氨酸trpoperon色氨酸Thetrpoperonencodesfivestructuralgenesrequiredfortryptophan(色氨酸)

synthesis.Thesegenesareregulatedtoefficientlyexpressonlywhentryptophan(Trp)islimiting.Twolayersofregulationareinvolved:(1)transcriptionrepressionbytheTrprepressor(阻遏作用)-粗调;(2)attenuation(衰减作用)-细调TranscriptionrepressionbytheTrprepressor(阻遏作用)-粗调低色氨酸:去阻遏高色氨酸:阻遏inactiverepressoractiverepressorNotranscriptionTrprepressorisencodedbyaseparateoperontrpR,andspecificallyinteractswiththeoperator

thatoverlapswiththepromotersequence

Therepressorcanonlybindtotheoperatorwhenitiscomplexedwithtryptophan

Therefore,tryptophanisa

corepressor(辅阻遏物)

andinhibitsitsownsynthesisthroughnegativefeed-backregulationlacIpromoterRNApolymeraselacZlacYlacANotranscriptiontranscriptionoperatoractiverepressorinactiverepressor阻遏去阻遏ControlelementStructuralgenesBasicprincipleofregulationofgeneexpression:Interactionbetweentrans-actingfactor(regulatoryprotein)andcis-actingelement(controlelement)BasicmechanismofregulationofgeneexpressionRepressor(阻遏作用):servesastheprimaryswitchtoregulatetheexpressionofgenesinthetrpoperon—粗调Attenuation(衰减作用):servesasthefineswitchtodetermineifthegenesneedtobeefficientlyexpressed—细调

Attenuation(衰减作用)DiscoveryofAttenuation(衰减作用)RegulatorymechanismofAttenuationTranscriptionofthetrpoperonisprematurallystoppedifthetryptophanlevelisnotlowenough,whichresultsintheproductionofaleaderRNAof161bp.(转录提前终止,WHY?)161bpleaderRNATrp-Trp+fromphenomenontomechanismScientificresearchmethod:Inductionofenzyme(酶的诱导现象)Addlactoseremovelactosetime/minβ-galactosidase(半乳糖苷酶)galactosidepermease(半乳糖苷渗透酶)β-galactosidasegeneNegativecontroloftheLacoperon:interactionbetweenrepressorandoperator(负调控机制)glucoseusedlactoseusedlagTheexpressionoflacoperonwasinhibitedbyglucosePhenomenon:二次生长曲线PositivecontroloftheLacoperon:interactionbetweenCAP-cAMPcomplexandCAPbindingsite(正调控机制)

Transcriptionofthetrpoperonisprematurallystoppedifthetryptophanlevelisnotlowenough,whichresultsintheproductionofaleaderRNAof161bp.(现象:转录提前终止,WHY?)161bpleaderRNATrp-Trp+色氨酸14个氨基酸的小肽34Rho-independentterminator(不依赖于因子的转录终止子)TranscriptionisterminatedbysignalswithintheRNAsequence(转录终止信号)Rho-independentterminator

containsashortinvertedrepeat(~20bp)andastretchof~8A:Tbasepairs

Weakestbasepairing:A:UmakethedissociationeasierFig12-10transcriptiontermination色氨酸14个氨基酸的前导肽1234234112Tryptophan:UGGTranscriptionandtranslationinbacteriaarecoupled(细菌体内的转录和翻译是偶联的).

Therefore,synthesisoftheleaderpeptideimmediatelyfollowsthetranscriptionofleaderRNA.Theleaderpeptide

containstwotryptophancodons.Ifthetryptophanlevelisverylow,theribosomewillpauseatthesesites.缺少色氨酸充足色氨酸形成不依赖于因子的转录终止子,转录终止不能够形成不依赖于因子的转录终止子,转录继续LowTrpHighTrpFig16-21Complementary2:3ElongationoftranscriptionComplementary3:4terminationoftranscriptionTranscriptionofthetrpoperonisprematurallystoppedifthetryptophanlevelisnotlowenough,whichresultsintheproductionofaleaderRNAof161bp.(现象:转录提前终止,WHY?)161bpleaderRNATrp-Trp+Summaryof

attenuationAtypicalnegativefeed-backregulationProvidesanexampleofregulationwithouttheuseofaregulatoryprotein,butusingRNAstructureinstead3.Attenuationalonecanproviderobustregulation:otheraminoacidsoperonslikehisandleu

havenorepressorsandrelyentirelyonattenuationfortheirregulationControlelementStructuralgenesBasicprincipleofregulationofgeneexpression:Interactionbetweentrans-actingfactor(regulatoryprotein)andcis-actingelement(controlelement)Furtherreading:TOPIC1

PrinciplesofTranscriptionalRegulationinprokaryotesTOPIC2RegulationofTranscriptionInitiation:ExamplesfromBacteria(Lacoperon)TOPIC3ExamplesofGeneRegulationafterTranscriptionInitiation(Trpoperon),attenuationSUMMARYTOPIC1PrinciplesofTranscriptionalRegulationGene

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