分子生物學(xué)-MajorShiftsinBacterialTranscription課件

8.1SigmaFactorSwitching8.2TheRNAPolymeraseEncodedinPhageT7

8.3InfectionofE.colibyPhageλ(antitermination)Chapter8

MajorShiftsinBacterialTranscription8.1SigmaFactorSwitchingPhageinfectionofbacteriumsubvertshosttranscriptionmachineryInprocess,establishesatime-dependentprogramoftranscriptionFirstearlyphagegenesaretranscribedThisisfollowedbythelatergenesLateintheinfectiouscyclethereisnolongertranscriptionofthehostgenes,onlyphagegenesChangeinwhatgenestranscribediscausedbyachangeintranscriptionmachinery,inRNApolymeraseitselfChapter6establishedthatσisthekeyfactorindeterminingspecificityofT4DNAtranscriptionToshiftthetranscriptionprocessσisalikelycandidateStudyoftheprocessdoneinB.subtilisanditsphage,SPO1PhageInfectionPolymeraseswereseparatedbychromatographyandsubjectedtoSDStodisplaytheirsubunits.EnzymeB(firstlane)containsthecoresubunits(β’,β,αandω),aswellassubunitIV(gp28).EnzymeC(secondlane)containsthecoresubunitsplussubunitsV(gp34)andVI(gp33).Thelasttwolanescontainseparatedσandδsubunits,respectively.gp28gp34gp33AThisswitchingisdirectedbyasetofphage-encodedσfactorsthatassociatewiththehostcoreRNApolymeraseTheseσfactorschangethehostpolymerasespecificityofpromoterrecognitionfromearlytomiddletolateThehostσfactorisspecificforthephageearlygenesPhagegp28proteinswitchesthespecificitytothemiddlegenesPhagegp33andgp34proteinsswitchtolatespecificityTranscriptionSwitchingSporulationDuringinfection,phageSPO1changesspecificityofhostRNApolymeraseSametypeofmechanismappliestochangesingeneexpressionduringsporulationBacteriacanexistindefinitelyinvegetativestateifnutrientsareavailableUnderstarvationconditions,B.subtilisformsendospores,toughdormantbodiesSporulationinvolvesthedifferentiationofavegetativebacteriumintoamothercellthatislysedandasporethatisreleased.8hrs,40%RNAspoOAH43PO4σFtriggerssynthesisofthenextsigmafactorintheforesporeandturnsonSpoIIRwhichcausesSpoIIGAtocleavepro-σE.SporulationSwitchingDuringsporulation,awholenewsetofgenesisturnedon,andvegetativegenesareturnedoffSwitchoccurslargelyattheleveloftranscriptionSeveralnews-factorsdisplacethevegetatives-factorfromthepolymerasecoreEachs-factorhasitsownpreferredpromotersequenceGeneswithMultiplePromoters

Somesporulationgenesmustbeexpressedduring2ormorephasesofsporulationwhendifferents-factorspredominateGenestranscribedunderdifferentconditionsareequippedwithtwodifferentpromotersEachpromoterisrecognizedbyoneoftwodifferents-factorsThisensurestheirexpressionnomatterwhichfactorispresentAllowsfordifferentialcontrolunderdifferentconditions?

TheB.subtilisspoVGGene

OneofthesporulationgeneswithtwopromotersisspoVG,whichistranscribedbybothEσBandEσE(holoenzymesbearingeitherσBorσE).ResultofheatshockontranscriptioninE.coli.Top:Atnormaltemperature,σH(green)issomehowsequesteredsoitcannotbindtothecorepolymerase.Asaresult,thenormalσA(blue)associateswithcore,directingittonormalvegetativepromoters(pA).Asaresult,vegetativetranscripts(blue)areproduced.Bottom:Onheating,theisolatedσHisreleased.ItcompeteswithσAforbindingtocoreanddirectsthepolymerasetoheatshockpromoterspH.Consequently,heatshocktranscripts(green)aremade.MapoftheDNAregionusedtoassaythetranscriptioninitiationfromtheE.coliglnAgene.TheboldredregioncontainstheglnApromotersglnAP1andglnAP2.Downstreamfromthesepromoters,intheblueregionoftheDNA,liestheT7phageterminator(tT7).TranscriptsinitiatingatglnAP1orglnAP2andterminatingattT7producetruncatedtranscriptsof415and300nt,respectively.?TheE.coliglnAGeneσ70σ54-NH4+strong-carbonweakPhagelikeT7,T3,and11havesmallgenomesandmanyfewergenesThesephagehave3phasesoftranscription:classesI,II,andIIIOf5classIgenes,gene1isnecessaryforclassIIandclassIIIgeneexpressionIfgene1ismutated,onlyclass1genesaretranscribedGene1codesforaphage-specificRNApolymeraseofjustonepolypeptide8.2TheRNAPolymeraseEncodedinPhageT7Gene1RNApolymerasetranscribesonlyT7classIIandIIIgenes,notclassIgenesRNApolymeraseofphageT7isunusuallyspecificThispolymerasewilltranscribevirtuallynoothernaturaltemplateAsimilarpolymerasehasbeenisolatedfromphageT3.ItisspecificforT3,ratherthanT7genes.Gene1RNAPolymerase8.3InfectionofE.colibyPhagelVirulentphagereplicateandkilltheirhostbylysingorbreakingitopenTemperatephage,suchasl,infectcellsbutdon’tnecessarilykillThetemperatephagehave2pathsofreproductionLyticmode:infectionprogressesasinavirulentphageLysogenicmode:phageDNAisintegratedintothehostgenomeλ

repressorcIgenelysogen

prophage

LysogenicPhaseCroTwoPathsofPhageReproduction12ntGeneticMapofPhagelDNAexistsinlinearforminthephageAfterinfectionofhostbeginsthephageDNAcircularizesThisispossibleasthelinearformhasstickyendsGeneticsitessurroundingtheNgeneinclude:Leftpromoter,PLOperator,OLTranscriptionterminatorWhenNispresent:NbindstranscriptofNutilizationsite(nutsite)InteractswithproteincomplexboundtopolymerasePolymeraseignoresnormaltranscriptionterminator,continuesintodelayedearlygenesNAntiterminationFunctionEffectofNonleftwardtranscriptionHostproteinsFiveproteinscollaborateinantiterminationatthelimmediateearlyterminatorsNusAandS10bindRNApolymeraseNandNusBbindtotheboxBandboxAregionsofthenutsiteNandNusBbindtoNusAandS10probablytetheringthetranscripttothepolymeraseNusAstimulatesterminationatintrinsicterminatorbyinterferingwithbindingbetweenupstreampartofterminatorhairpinandcorepolymeraseProteinsInvolvedinN-DirectedAntiterminationProteincomplexesinvolvedinN-directedantiterminationWeak,nonprocessivecomplexStrong,processivecomplex???InvitroInvitro(invitro)ModelfortheFunctionofNusAandNinIntrinsicTerminationDemonstrationofprotein-RNAcontactsinthepausedEC,withandwithoutNusAandNAntiterminationandQAntiterminationinthellateregionrequiresQQbindstotheQ-bindingregionofthequtsiteasRNApolymeraseisstalledjustdownstreamoflatepromoterBindingofQtothepolymeraseappearstoaltertheenzymesoitcanignoretheterminatorandtranscribethelategenesMapofthePR’regionoftheλgenome.ThePR’promotercomprisesthe–10and–35boxes.ThequtsiteoverlapsthepromoterandincludestheQbindingsiteupstreamofthe–10box,thepausesignaldownstreamofthetranscriptionstartsite,andthepausesiteatpositions+16and+17.A27-kDphageprotein(lrepressor,CI)appearsandbindsto2phageoperatorregionsCIshutsdowntranscriptionofallgenesexceptforcI,geneforlrepressoritselfLysogenisabacteriumharboringintegratedphageDNAThisintegratedDNAiscalledaprophageLysogenicModeEstablishingLysogenyPhageestablishlysogenyby:CausingproductionofrepressortobindtoearlyoperatorsPreventingfurtherearlyRNAsynthesisDelayedearlygeneproductsareusedIntegrationintothehostgenomeProductsofcIIandcIIIallowtranscriptionofthecIgeneandproductionoflrepressorPromotertoestablishlysogenyisPREModelofEstablishingLysogenyDelayedearlytranscriptionfromPRproducescIImRNAtranslatedtoCIICIIallowsRNApolymerasetobindtoPREandtranscribethecIgene,resultinginrepressor??PhageλestablisheslysogenybycausingproductionofenoughrepressortobindtotheearlyoperatorsandpreventfurtherearlyRNAsynthesis.ThepromoterusedforestablishmentoflysogenyisPRE,whichliestotherightofPRandcro.TranscriptionfromthispromotergoesleftwardthroughthecIgene.TheproductsofthedelayedearlygenescIIandcIII

alsoparticipateinthisprocess:CII,bydirectlystimulatingpolymerasebindingtoPREandPI;CIII,byslowingdegradationofCII.SUMMARYAutoregulationofthecIGeneduringLysogenyAslrepressorappears,bindsasadimertoloperators,ORandOLresultsin:RepressorturnsofffurtherearlytranscriptionInterruptslyticcycleTurnoffofcroveryimportantasproductCroactstocounter（對抗）

repressoractivityStimulatesownsynthesisbyactivatingPRMMaintainingLysogenyPREThreeoftheprecedingassertions:1)theλrepressoratlowconcentrationcanstimulatetranscriptionofitsowngene2)therepressorathighconcentrationcaninhibittranscriptionofitsowngene3)therepressorcaninhibitcrotranscriptionMapoftheDNAfragmentusedtoassaytranscriptionfromcIandcropromoters.TheredarrowsdenotetheinvitrocIandcrotranscripts,someofwhichterminateprematurely110nt300ntTherepressorclearlyinhibitedcrotranscription,butitgreatlystimulatedcItranscriptionatlowconcentration,theninhibitedcItranscriptionathighconcentration.RepressorproteinAdimerof2identicalsubunitsEachsubunithas2domainswithdistinctrolesAmino-terminalistheDNA-bindingendofmoleculeCarboxyl-terminalissiteofrepressor-repressorinteractionthatmakesdimerizationandcooperativebindingpossibleRepressorProteinModelofInvolvementofOLinRepression（阻遏）ofPRandPRMInvolvementofOLinRepressionRepressorbindstoOR1andOR2cooperatively,butleavesOR3RNApolymerasetoPRMwhichoverlapsOR3insuchawayitcontactsrepressorboundtoOR2Protein-proteininteractionisrequiredforpromotertoworkefficientlyHighlevelsofrepressorcanrepresstranscriptionfromPRMProcessmayinvolveinteractionofrepressordimersboundtoOR1,OR2,andOR3RepressordimersboundtoOL1,OL2,andOL3viaDNAloopingRNAPolymerase/RepressorInteraction?PrincipleofIntergenicSuppressionDirectinteractionbetweenrepressorandpolymeraseisnecessaryforefficienttranscriptionfromPRMMutantwithcompensatingaminoacidchangeinRNApolymerasesubunitrestoresinteractionwithmutantrepressorInintergenicsuppression,amutantinonegenesuppressesamutationinanotherSelectionforintergenicsuppressorofλ

cIpcmutation(Arg596His)wild-typemutantversions-irrelevantmutationActivationViaSigmaPromoterssubjecttopolymerase-repressoractivationhaveweak-35boxesTheseboxesarepoorlyrecognizedbysActivatorsiteoverlaps-35box,placesactivatorinpositiontointeractwithregion4IntergenicsuppressormutationstudiesshowthatcrucialinteractionbetweenrepressorandRNApolymeraseinvolvesregion4ofthes-subunitofthepolymerasePolypeptidebindsneartheweak-35boxofPRMplacingthes-region4closetotherepressorboundtoOR2Repressorcaninteractwiths-factorhelpingtocompensateforweakpromoterOR2servesasanactivatorsiteRepressorClisanactivatoroftranscriptionfromPRMSUMMARYBalancebetweenlysisorlysogenyisdelicatePlacephageparticlesonbacteriallawnIflyticinfectionoccursProgenyspreadandinfectothercellsCircularholeseeninlawniscalledplaqueInfection100%lyticgivesclearplaquePlaquesoflareusuallyturbidmeaninglivelysogenispresentSomeinfectedcellssufferthelyticcycle,othersarelysogenizedDeterminingtheFateofalInfectionWhatdetermineswhetheragivencellinfectedbyλwillenterthelyticcycleorlysogeny??Thebalancebetweenthesetwofatesisdelicate,andweusuallycannotpredicttheactualpathtakeninagivencell.?Thecellswithinaplaqueareallgeneticallyidentical,andsoarethephages,sothechoiceoffateisnotgenetic.

Instead,itseemstorepresentaracebetweentheproductsoftwogenes:cIandcro.

BattleBetweencIandcroThecIgenecodesforrepressor,blocksOR1,OR2,OL1,andOL2soturningoffearlytranscriptionThisleadstolysogenyThecrogenecodesforCrothatblocksOR3andOL3,turningoffcItranscriptionThisleadstolyticinfectionGeneproductinhighconcentrationfirstdeterminescellfateWhatdetermineswhethercIorcrowinstherace?ThemostimportantfactorseemstobetheconcentrationofthecIIgeneproduct,CII.ItactivatesPREandhelpsturnonthelysogenyprogram.WhatcontrolstheconcentrationofCII?CIIIprotectsCIIagainstcellularproteases,buthighproteaseconcentrationscanoverwhelmCIII,destroyCII,andensurethattheinfectionwillbelytic.Suchhighproteaseconcentrationsoccurundergoodenvironmentalconditionsrichmedium,forexa