分子生物学英文课件 (8).ppt

WelcomeEachofYoutoMyMolecularBiologyClass 2 MolecularBiologyoftheGene 5 E Watsonetal 2004 PartI ChemistryandGeneticsPartII MaintenanceoftheGenomePartIII ExpressionoftheGenomePartIV RegulationPartV Methods 2005 5 10 3 PartIVRegulation Ch16 RegulationinprokaryotesCh17 RegulationineukaryotesCh18 Regulationduringdevelopmentandindiseases briefintroduction Ch19 Comparativegenomicsandevolutionofanimaldiversity Notcoveredinthelecture 4 Housekeepinggenes expressedconstitutively essentialforbasicprocessesinvolvingincellreplicationandgrowth Induciblegenes expressedonlywhentheyareactivatedbyinducersorcellularfactors Expressionofmanygenesincellsareregulated 5 Chapter16RegulationprinciplesandHowgenesareregulatedinbacteriaChapter17BasicmechanismofgeneexpressionineukaryotesChapter18ThemechanismofRNAiandtheroleofmiRNAindevelopmentandcancergenesis 6 SurfingthecontentsofPartIV Theheartofthefrontierbiologicaldisciplines 7 Someofthepeopleswhosignificantlycontributetotheknowledgeofgeneregulation 8 9 10 Chapter16GeneRegulationinProkaryotes MolecularBiologyCourse 11 TOPIC1PrinciplesofTranscriptionalRegulation watchtheanimation TOPIC2RegulationofTranscriptionInitiation ExamplesfromBacteria Lacoperon alternativesfactors NtrC MerR Galrep araBADoperon TOPIC3ExamplesofGeneRegulationafterTranscriptionInitiation Trpoperon TOPIC4TheCaseofPhage LayersofRegulation 12 Topic1 PrinciplesofTranscriptionRegulation CHAPTER16GeneRegulationinProkaryotes 5 10 2005 13 1 GeneExpressionisControlledbyRegulatoryProteins 调控蛋白 GeneexpressionisveryoftencontrolledbyExtracellularSignals whicharecommunicatedtogenesbyregulatoryproteins PositiveregulatorsoractivatorsINCREASEthetranscriptionNegativeregulatorsorrepressorsDECREASEorELIMINATEthetranscription PrinciplesofTranscriptionRegulation 14 2 Geneexpressioniscontrolledatdifferentstages 基因表达可以发生在不同时期 PrinciplesofTranscriptionRegulation Thebulkofgeneregulationtakesplaceattheinitiationoftranscription Someinvolvetranscriptionalelongation termination RNAprocessing andtranslationofthemRNAintoprotein 15 Fig12 3 initiation PromoterBinding closedcomplex Promoter melting opencomplex Promoterescape Initialtranscription 16 Fig12 3 Elongationandtermination Termination Elongation 17 3 Targetingpromoterbinding manypromotersareregulatedbyactivators 激活蛋白 thathelpRNAPbindDNA recruitment andbyrepressors 阻遏蛋白 thatblockthebinding RNAPbindsmanypromotersweakly activatorsthatcontaintwobindingsitestobindaDNAsequenceandRNAPsimultaneouslycanenhancetheRNAPaffinitywiththepromoters andthusincreasesgenetranscription Thisiscalledrecruitmentregulation 招募调控 Onthecontrary Repressorscanbindtotheoperatorinsideofthepromoterregion whichpreventsRNAPbindingandthetranscriptionofthetargetgene PrinciplesofTranscriptionRegulation 18 a AbsenceofRegulatoryProteins basallevelexpression b Repressorbindingtotheoperatorrepressesexpression c Activatorbindingactivatesexpression Fig16 1 19 4Targetingtransitiontotheopencomplex Allosteryregulation 异构调控 aftertheRNAPolymeraseBinding PrinciplesofTranscriptionRegulation Insomecases RNAPbindsthepromotersefficiently butnospontaneousisomerizationoccurstoleadtotheopencomplex resultinginnoorlowtranscription Someactivatorscanbindtotheclosedcomplex inducingconformationalchangeineitherRNAPorDNApromoter whichconvertstheclosedcomplextoopencomplexandthuspromotesthetranscription 20 Fig16 2 Allosteryregulation Allosteryisnotonlyamechanismofgeneactivation itisalsooftenthewaythatregulatorsarecontrolledbytheirspecificsignals 21 5Targetingpromoterescapebysomerepressors PrinciplesofTranscriptionRegulation Repressorscanworkinways blockingthepromoterbinding blockingthetransitiontotheopencomplex blockingpromoterescape 22 Somepromotersareinefficientatmorethanonestepandcanbeactivatedbymorethanonemechanism Activationmechanismsincluderecruitment 招募 andallostery 异构 23 6 Cooperativebinding recruitment andallosteryhavemanyrolesingeneregulation Forexample groupofregulatorsoftenbindDNAcooperatively activatorsand orrepressorsinteractwitheachotherandwiththeDNA helpingeachothertobindnearagenetheyregulated producesensitiveswitchestorapidlyturnonageneexpression integratesignals somegenesareactivatedwhenmultiplesignalsarepresent PrinciplesofTranscriptionRegulation 24 7 ActionataDistanceandDNALooping TheregulatorproteinscanfunctionevenbindingataDNAsitefarawayfromthepromoterregion throughprotein proteininteractionandDNAlooping Fig16 3 PrinciplesofTranscriptionRegulation 25 Fig16 4DNA bindingproteincanfacilitateinteractionbetweenDNA bindingproteinsatadistance Fig16 4 26 Topic2 RegulationofTranscriptionInitiation ExamplesfromBacteria CHAPTER16GeneRegulationinProkaryotes 27 Operon aunitofprokarytoicgeneexpressionandregulationwhichtypicallyincludes 1 Structuralgenesforenzymesinaspecificbiosyntheticpathwaywhoseexpressioniscoordinatelycontrolled 2 Controlelements suchasoperatorsequence 3 Regulatorgene s whoseproductsrecognizethecontrolelements Sometimesareencodedbythegeneunderthecontrolofadifferentpromoter 28 Controlelement Structuralgenes 29 Firstexample Lacoperon RegulationofTranscriptionInitiationinBacteria ThelactoseOperon 乳糖操纵子 30 TheenzymesencodedbylacZ lacY lacAarerequiredfortheuseoflactoseasacarbonsource Thesegenesareonlytranscribedatahighlevelwhenlactoseisavailableasthesolecarbonsource Fig16 5 TheLACoperon 1 Lactoseoperoncontainsaregulatorygeneand3structuralgenes and2controlelements 31 lacY encodesacellmembraneproteincalledlactosepermease 半乳糖苷渗透酶 totransportLactoseacrossthecellwall lacZ codesfor galactosidase 半乳糖苷酶 forlactosehydrolysis lacA encodesathiogalactosidetransacetylase 硫代半乳糖苷转乙酰酶 togetridofthetoxicthiogalacosides TheLACoperon 32 ThelacZ lacY lacAgenesaretranscribedintoasinglelacZYAmRNA polycistronicmRNA underthecontrolofasignalpromoterPlac LacZYAtranscriptionunitcontainsanoperatorsiteOlac positionbetweenbases 5and 21atthe3 endofPlac Bindswiththelacrepressor TheLACoperon 33 2 AnactivatorandarepressortogethercontroltheLacoperonexpression Theactivator CAP CataboliteActivatorProtein 代谢产物激活蛋白 orCRP cAMPReceptorProtein cAMP受体蛋白 responsestotheglucoselevel Therepressor lacrepressorthatisencodedbyLacIgene responsestothelactose Sugarswitch offmechanism TheLACoperon 34 Fig16 6 TheLACoperon 35 TheLACoperon Thesiteboundbylacrepressoriscalledthelacoperator Olac andtheOlacoverlapspromoter Plac ThereforerepressorboundtotheoperatorphysicallypreventsRNApolymerasefrombindingtothepromoter 3 LacrepressorboundtotheoperatorpreventsRNAPfrombindingtothepromoter TheLACoperon 36 Fig16 8 TheLACoperon 37 TheLACoperon CAPhastwobindingsites oneinteractswiththeCAPsiteontheDNAnearpromoter andoneinteractswithRNAP ThiscooperativebindingensuresthatRNAPeffectivelybindstoPlacandinitiatestranscriptionofLacZYA 4 CAPactivatestheLactranscriptionthroughrecruitmentofRNAPtotheweakPlac TheLACoperon 38 CAPsitehasthesimilarstructureastheoperator whichis60bpupstreamofthestartsiteoftranscription CAPalsointeractswiththeRNAPandrecruitittothepromoter Fig16 9 aCTD C terminaldomainoftheasubunitofRNAP 39 CAPbindsasadimer aCTD Fig16 10 CAPhasseparateactivatingandDNA bindingsurface TheLACoperon 40 5 CAPandLacrepressorbindDNAusingacommonstructuralmotif helix turn helixmotif TheLACoperon Fig16 11 OneistherecognitionhelixthatcanfitsintothemajorgrooveoftheDNA 41 DNAbindingbyahelix turn helixmotif Fig16 12HydrogenBondsbetweenlrepressorandthemajorgrooveoftheoperator 42 Lacoperoncontainsthreeoperators theprimaryoperatorandtwootheroperatorslocated400bpdownstreamand90bpupstream Lacrepressorbindsasatetramer 四聚体 witheachoperatoriscontactedbyarepressordimer 二聚体 respectively Fig16 13 43 6TheactivityofLacrepressorandCAParecontrolledallostericallybytheirsignals Lactoseisconvertedtoallolactosebyb galactosidase thereforelactosecanindirectlyturnofftherepressor GlucoselowersthecellularcAMPlevel therefore glucoseindirectlyturnoffCAP TheLACoperon Allolactose turnofLacrepressorcAMP turnonCAP 44 i p o z y a b Galactosidase Permease Transacetylase Presenceoflactose Inactive Lackofinducer thelacrepressorblockallbutaverylowleveloftrans criptionoflacZYA WhenLactoseispresent thelowbasallevelofpermeaseallowsitsuptake andb galactosidasecatalyzestheconversionofsomelactosetoallolactose Allolactoseactsasaninducer bindingtothelacrepressorandinactivateit Responsetolactose 45 Responsetoglucose 46 Aregulator CAP workstogetherwithdifferentrepressoratdifferentgenes thisisanexampleofCombinatorialControl Infact CAPactsatmorethan100genesinE coli workingwithanarrayofpartners 7 CombinatorialControl 组合调控 CAPcontrolsothergenesaswell 47 Secondexample Alternativesfactor RegulationofTranscriptionInitiationinBacteria Alternativesfactor 可变s因子 directRNApolymerasetoalternativesiteofpromoters 48 factorsubunitboundtoRNApolymerasefortranscriptioninitiation Ch12 49 Different factorsbindingtothesameRNAP conferringeachofthemanewpromoterspecificity 70factorsismostcommononeinE coliunderthenormalgrowthcondition 50 Manybacteriaproducealternativesetsof factorstomeettheregulationrequirementsoftranscriptionundernormalandextremegrowthcondition Bacteriophagehasitsown factors E coli Heatshock 32 SporulationinBacillussubtilis Bacteriophage factors 51 Heatshock 热休克 Around17proteinsarespecificallyexpressedinE coliwhenthetemperatureisincreasedabove37 C TheseproteinsareexpressedthroughtranscriptionbyRNApolymeraseusinganalternative factor 32codedbyrhoHgene 32hasitsownspecificpromoterconsensussequences Alternativesfactors 52 Manybacteriophagessynthesizetheirown factorstoendowthehostRNApolymerasewithadifferentpromoterspecificityandhencetoselectivelyexpresstheirownphagegenes Bacteriophages Alternativesfactors 53 B subtilisSPO1phageexpressesacascadeof factorswhichallowadefinedsequenceofexpressionofdifferentphagegenes Fig16 14 Alternativesfactors 54 Thirdexample NtrCandMerRandallostericactivation RegulationofTranscriptionInitiationinBacteria TranscriptionalactivatorsNtrCandMerRworkbyallosteryratherthanbyrecruitment 55 ReviewThemajorityofactivatorsworkbyrecruitment suchasCAP TheseactivatorssimplybringanactiveformofRNApolymerasetothepromoterInthecaseofallostericactivation RNAPinitiallybindsthepromoterinaninactivecomplex andtheactivatortriggersanallostericchangeinthatcomplextoactivatetranscription 56 IntheabsenceofNtrCandMerR RNAPbindstothecorrespondingpromotertoformaclosedstablecomplex NtrCactivatorinducesaconformationalchangeintheenzyme triggeringtransitiontotheopencomplexMerRactivatorcausestheallostericeffectontheDNAandtriggersthetransitiontotheopencomplex 57 1 NtrChasATPaseactivityandworksfromDNAsitesfarfromthegene NtrCandMerRandallostericactivation NtrCcontrolsexpressionofgenesinvolvedinnitrogenmetabolism 氮代谢 suchastheglnAgeneNtrChasseparateactivatingandDNA bindingdomains andbindsDNAonlywhenthenitrogenlevelsarelow 58 Lownitrogenlevels 低水平氮 NtrCphosphorylationandconformationalchange NtrC bindsDNAsitesat 150positioasadimer NtrC interactswith 54 glnApromoterrecognition NtrCATPaseactivityprovidesenergyneededtoinduceaconformationchangeinpolymerase transcriptionSTARTs Fig16 15activationbyNtrC 59 2 MerRactivatestranscriptionbytwistingpromoterDNA NtrCandMerRandallostericactivation MerRcontrolsagenecalledmerT whichencodesanenzymethatmakescellsresistanttothetoxiceffectsofmercury 抗汞酶 Inthepresenceofmercury 汞 MerRbindstoasequencebetween 10and 35regionsofthemerTpromoterandactivatesmerTexpression 60 Asa 70promoter merTcontains19bpbetween 10and 35elements thetypicallengthis15 17bp leavingthesetwoelementsrecognizedby 70neitheroptimallyseparatednoraligned 61 Fig16 15StructureofamerT likepromoter 62 Fig16 15 WhenHg2 isabsent MerRbindstothepromoterandlocksitintheunfavorableconformationWhenHg2 ispresent MerRbindsHg2 andundergoesconformationalchange whichtwiststhepromotertorestoreittothestructureclosetoastrong 70promoter 63 Repressorsworkinmanyways reviewBlockingRNApolymerasebindingthroughbindingtoasiteoverlappingthepromoter LacrepressorBlockingthetransitionfromtheclosedtoopencomplex Repressorsbindtositesbesideapromoter interactwithpolymeraseboundatthatpromoterandinhibitinitiation E coliGalrepressorBlockingthepromoterescape P4proteininteractionwithPA2c bacteriophagef29 64 Fourthexample araBADoperon RegulationofTranscriptionInitiationinBacteria 65 1 AraCandcontrolofthearaBADoperonbyanti activation ThearaBADoperon ThepromoterofthearaBADoperonfromE coliisactivatedinthepresenceofarabinose 阿拉伯糖 andtheabsenceofglucoseanddirectsexpressionofgenesencodingenzymesrequiredforarabinosemetabolism ThisisverysimilartotheLacoperon 66 DifferentfromtheLacoperon twoactivatorsAraCandCAPworktogethertoactivatethearaBADoperonexpression Fig16 18 CAPsite 194bp 67 BecausethemagnitudeofinductionofthearaBADpromoterbyarabinoseisverylarge thepromoterisoftenusedinexpressionvector IffusingagenetothearaBADpromoter theexpressionofthegenecanbeeasilycontrolledbyadditionofarabinose 阿拉伯糖 Whatisanexpressionvector TheanswerisintheMethodspart 68 Topic3 ExamplesofGeneRegulationatStepsAfterTranscriptionInitiation CHAPTER16GeneRegulationinProkaryotes 69 Firstexample thetryptophanoperon 色胺酸操纵子 ExamplesofGeneRegulationatStepsAfterTranscriptionInitiation 70 1 Aminoacidbiosyntheticoperonsarecontrolledbyprematuretranscriptiontermination thetrpoperon 71 Thetrpoperonencodesfivestructuralgenesrequiredfortryptophan 色胺酸 synthesis Thesegenesareregulatedtoefficientlyexpressonlywhentryptophanislimiting Twolayersofregulationareinvolved 1 transcriptionrepressionbytheTrprepressor initiation 2 attenuation TheTRPoperon 72 TheTrprepressor 色氨酸阻遏物 TheTRPoperon 73 TrprepressorisencodedbyaseparateoperontrpR andspecificallyinteractswiththeoperatorthatoverlapswiththepromotersequenceTherepressorcanonlybindtotheoperatorwhenitiscomplexedwithtryptophan Therefore Tryisaco repressorandinhibitsitsownsynthesisthroughend productinhibition negativefeed backregulation TheTRPoperon Rememberthelacrepressoractsasaninducer 74 TheTRPoperon Therepressorreducestranscriptioninitiationbyaround70 fold whichismuchsmallerthanthebindingoflacrepressor TherepressorisadimeroftwosubunitswhichhasastructurewithacentralcoreandtwoflexibleDNA readingheads carboxyl terminalofeachsubunit 75 trpRoperon trpoperon TheTRPoperon 76 Attenuation 衰减作用 aregulationatthetranscriptionterminationstep asecondmechanismtoconfirmthatlittletryptophanisavailable TheTRPoperon 77 RepressorservesastheprimaryswitchtoregulatetheexpressionofgenesinthetrpoperonAttenuationservesasthefineswitchtodetermineifthegenesneedtobeefficientlyexpressed 78 Fig16 19 Transcriptionofthetrpoperonisprematurallystoppedifthetryptophanlevelisnotlowenough whichresultsintheproductionofaleaderRNAof161nt WHY 79 Transcriptionandtranslationinbacteriaarecoupled 细菌体内的转录和翻译是偶联的 Therefore synthesisoftheleaderpeptideimmediatelyfollowsthetranscriptionofleaderRNA Theleaderpeptidecontainstwotryptophancodons Ifthetryptophanlevelisverylow theribosomewillpauseatthesesites RibosomepauseatthesesitesalterthesecondarystructureoftheleaderRNA whicheliminatestheintrinsicterminatorstructureandallowthesuccessfultranscriptionofthetrpoperon 80 Fig16 20TheleaderRNAandleaderpeptideofthetrpoperon 81 LowTrp HighTrp Fig16 21 Complementary2 3Elongationoftranscription Complementary3 4terminationoftranscription 82 Importanceofattenuation Atypicalnegativefeed backregulationUseofbothrepressionandattenuationallowsafinetuningoftheleveloftheintracellulartryptophan Attenuationalonecanproviderobustregulation otheraminoacidsoperonslikehisandleuhavenorepressorsandrelyentirelyonattenuationfortheirregulation Providesanexampleofregulationwithouttheuseofaregulatoryprotein butusingRNAstructureinstead 83 Secondexample Riboswitches aRNAstructurecontrolmechanism ExamplesofGeneRegulationatStepsAfterTranscriptionInitiation RiboswitchesareregulatoryRNAelementsthatactasdirectsensorsofsmallmoleculemetabolitestocontrolgenetranscriptionortranslation 84 Box4 RiboswitchesoperatingattheleveloftranscriptionterminationusinganAntiterminationmechanism Riboswitchesoperatingattheleveloftranslation controllingtheformationofanRNAstructurethatmaskstheribosomebindingsiteonmRNA 85 Tucker1andBreaker CurrentOpinioninStructuralBiology2005 15 342 348 代谢物 代谢物