分子生物學(xué)課件：ch16 Gene Regulation in Prokaryotes

1Chapter16

GeneRegulationinProkaryotesPrinciplesofTranscriptionalRegulationRegulationofTranscriptionInitiation:

ExamplesfromBacteria:Lac

operonalternativesfactors,

NtrC,MerR,Galrep

araBAD

operonExamplesofGeneRegulationafterTranscriptionInitiationTheCaseofPhageλ:LayersofRegulationTranscriptioninitiationElongationandtermination:

AntiterminationandbeyondPrinciplesoftranscriptionalregulationRegulatoryproteinsactivators:helpRNApolymerasebindDNAafterRNApolymerasebindingRepressors:blockthatbinding

othersMechanism:Cooperativebinding

allostery

ActionatadistanceandDNAloopingGeneExpressionisControlledbyRegulatoryProteinsGeneexpressionisveryoftencontrolledbyExtracellularSignals,whicharecommunicatedtogenesbyregulatoryproteins

:PositiveregulatorsoractivatorsincreasethetranscriptionNegativeregulatorsorrepressorsdecreaseoreliminatethetranscriptiona.AbsenceofRegulatoryProteins(operator)b.ToControlExpressionc.ToActivateExpressionFig16-1Recruitment:cooperativebindingofproteinstoDNARegulatoryproteinsactivators:helpRNApolymerasebindDNA

afterRNApolymerasebindingRepressors:blockthatbinding

othersMechanism:Cooperativebinding

allostery

ActionatadistanceandDNAloopingAllostery

RegulateStepsafterRNAPolymeraseBindingTargetingtransitiontotheopencomplexExamples:ActivatorpromoterNtrC

glnAMerR

merTSomepromotersareinefficientatmorethanonestepandcanbeactivatedbymorethanonemechanismRepressorscanworkinwaysotherthanjustblockingthepromoterbinding.Forexample,inhibitionofthetransitiontotheopencomplex.Regulatoryproteinsactivators:helpRNApolymerasebindDNA

afterRNApolymerasebindingRepressors:blockthatbinding

othersMechanism:Cooperativebinding

allostery

ActionatadistanceandDNAloopingActionataDistanceandDNALooping.

SomeproteinsinteractwitheachotherevenwhenboundtositeswellseparatedontheDNADNA-bindingproteincanfacilitateinteractionbetweenDNA-bindingproteinsatadistanceTargetingterminationandbeyond:AntiterminationandBeyond

Thebulkofgeneregulationtakesplaceattheinitiationoftranscription.Someinvolvetranscriptionalelongation/termination,RNAprocessing,andtranslationofthemRNAintoprotein.PrinciplesofTranscriptionalRegulationRegulationofTranscriptionInitiation:

ExamplesfromBacteria:Lac

operonalternativesfactors,

NtrC,MerR,Galrep

araBAD

operonExamplesofGeneRegulationafterTranscriptionInitiation:(Trp

operon)TheCaseofPhageλ:LayersofRegulationOperon:

aunitofprokarytoicgeneexpressionandregulationwhichtypicallyincludes:

1.

Structuralgenes

forenzymesinaspecificbiosyntheticpathwaywhoseexpressioniscoordinatelycontrolled.

2.

Controlelements,suchasoperatorsequence.

3.

Regulatorgene(s)

whoseproductsrecognizethecontrolelements.SometimesareencodedbythegeneunderthecontrolofadifferentpromoterFirstexample:

LactoseoperonFirstexample:

Lactoseoperon1.AnactivatorandarepressortogethercontrolthelacgenesTheactivator:CAP(CataboliteActivatorProtein)orCRP(cAMPReceptorProtein);responsestotheglucoselevel.Therepressor:lacrepressorthatisencodedbyLacIgene;responsestothelactose.CAPandlacrepressorhaveopposingeffectsonRNApolymerasebindingtothelacpromoter

Thesiteboundbylacrepressoriscalledthe

lacoperator.

Thelacoperator

overlapspromoter,andsorepressorboundtotheoperatorphysicallypreventsRNApolymerasefrombindingtothepromoter.CAPbindstoasitewiththesimilarstructureastheoperator,whichis60bp

upstreamofthestartsiteoftranscription.CAPalsointeractswiththeenzymeandrecruitittothepromoter.

aCTD:C-terminaldomainoftheasubunitofRNAPCAPhasseparateactivatingandDNA-bindingsurfaceCAPbindsasadimeraCTDFig16-10CAPandlacrepressorbindDNAusingacommonstructuralmotif

BothCAPandlacrepressorbindDNAusingahelix-turn-helixmotif.OneistherecognitionhelixthatcanfitsintothemajorgrooveoftheDNA.DNAbindingbyahelix-turn-helixmotifFig16-12HydrogenBondsbetweenlrepressorandthemajorgrooveoftheoperatorLacrepressorbindsasatetramer,witheachoperatoriscontactedbyarepressordimer.Inadditiontotheprimaryoperator,therearetwootherlacoperatorslocated400bpdownstreamand90bpupstream,respectively.

Notallthebindingusea

helix-turn-helixmotifTheactivityofLacrepressorandCAParecontrolledallostericallybytheirsignalsBindingofthecorrespondingsignalsalterthestructureofthesetworegulatoryproteinsPolymerasebindingtopromoterisenhancedbyactiveCAPVerylowtranscriptionLowtranscriptionmaximumtranscriptionAregulator(CAP)workstogetherwithdifferentrepressoratdifferentgenes,thisisanexampleofCombinatorialControl.Infact,CAPactsatmorethan100genesinE.coli,workingwithanarrayofpartners.2.CombinatorialControl(組合調(diào)控):CAPcontrolsothergenesaswellRegulatoryproteinsActivators:Repressors:Cooperativebinding:allostery:

blockthatbinding

othersLactoseoperonlacrepressorTheactivityofLacrepressorandCAPCAPallosteryPromoterrecognition:

AlternativesfactordirectRNApolymerasetoalternativesiteofpromotersSecondexample:

AlternativesfactorDifferentσfactorsbindingtothesameRNAPol

ConfereachofthemanewpromoterspecificityManybacteriaproducealternativesetsofσfactorstomeettheregulationrequirementsoftranscriptionundernormalandextremegrowthconditionE.coli:Heatshock32

SporulationinBacillussubtilisBacteriophage

σfactorsHeatshock

Around17proteinsarespecificallyexpressedinE.coliwhenthetemperatureisincreasedabove37oC.TheseproteinsareexpressedthroughtranscriptionbyRNApolymeraseusinganalternativefactor32codedbyrhoHgene.32

hasitsownspecificpromoterconsensussequences.Manybacteriophagessynthesizetheirownσfactors

toendowthehostRNApolymerasewithadifferentpromoterspecificityandhencetoselectivelyexpresstheirownphagegenes.

Bacteriophagesphageexpressesacascadeofσfactorswhichallowadefinedsequenceofexpressionofdifferentphagegenes.ExpressearlygenesEncodeσ28Encodeσfactor

fortranscriptionoflategenesExpressmiddlegenesEncodeσ34Thirdexample:

NtrCandMerRand

allostericactivationNtrCandMerR:TranscriptionalactivatorsthatworkbyallosteryratherthanbyrecruitmentNtrCactivatorinducesaconformationalchangeintheenzyme,triggeringtransitiontotheopencomplexMerRactivatorcausestheallostericeffectontheDNAandtriggersthetransitiontotheopencomplexNtrChasATPaseactivityandworksfromDNAsitesfarfromthegeneNtrCcontrolsexpressionofgenesinvolvedinnitrogenmetabolism,suchastheglnAgeneNtrChasseparateactivatingandDNA-bindingdomains,andbindsDNAonlywhenthenitrogenlevelsarelow.LownitrogenlevelsNtrC

phosphorylationandconformationalchangeTheDNAbindingdomainbindsDNAsitesat~-150positionNtrCinteractswith54(glnApromoterrecognition)ATPhydrolysisandconformationchangeinpolymerase

transcriptionSTARTsMerRactivatestranscriptionbytwistingpromoterDNAMerRcontrolsagenecalledmerT,whichencodesanenzymethatmakescellsresistanttothetoxiceffectsofmercuryInthepresenceofmercury,MerRbindstoasequencebetween–10and–35regionsofthemerTpromoterandactivatesmerTexpression.Asa70promoter,merTcontains19bpbetween–10and–35elements(thetypicallengthis15-17bp),leavingthesetwoelementsneitheroptimallyseparatednoraligned.StructureofamerT-moterwitha17bpspacer.Hg2+Hg2+:

MerRbindstothepromoterandlocksitintheunfavorableconformation+Hg2+:MerRbindsHg2+andundergoconformationalchange,whichtwiststhepromotertorestoreittothestructureclosetoastrong70promoterBlockingRNApolymerasebindingthroughbindingtoasiteoverlappingthepromoter.LacrepressorBlockingthetransitionfromtheclosedtoopencomplex.Repressorsbindtositesbesideapromoter,interactwithpolymeraseboundatthatpromoterandinhibitinitiation.E.coliGalrepressorLockingthepromoterinaconformationincompatiblewithtranscriptioninitiation

P4proteinfromabacteriophageRepressorsworkinmanyways:SomerepressorsholdRNApolymeraseatthepromoterratherthanexcludingitFourthexample:條件

表達(dá)有Glu有GalP2啟動(dòng)S2開始轉(zhuǎn)錄galE，組成型表達(dá)有Glu無GalOE和OI相互作用，成環(huán)，轉(zhuǎn)錄只進(jìn)行20堿基便停止無Glu無GalP1不啟動(dòng)無Glu有GalP1啟動(dòng),3個(gè)基因轉(zhuǎn)錄

AraCandcontrolofthearaBAD

operonbyantiactivationThepromoterofthearaBAD

operonfromE.coliisactivatedinthepresenceofarabinose(阿拉伯糖)andtheabsenceofglucoseanddirectsexpressionofgenesencodingenzymesrequiredforarabinosemetabolism.ThisisverysimilartotheLacoperon.

Fifthexample:

araBAD

operonDifferentfromtheLacoperon,twoactivatorsAraCandCAPworktogethertoactivatethearaBAD

operonexpressionFunctionofAraCinregulatinggeneexpressionatthearapromoterpBADRegionsofcontactRNAPaAraC

dimerRegulatoryproteinsActivators:Repressors:Cooperativebinding:allostery:

blockthatbindingBlockingthetransitiontoopencomplex

Lockingthepromoter

lacrepressorTheactivityofLacrepressorandCAPCAPNtrCandMerRGalrepressorP4proteinfromabacteriophageCombinatorialControlAraCAraCPrinciplesofTranscriptionalRegulationRegulationofTranscriptionInitiation:

ExamplesfromBacteria

ExamplesofGeneRegulationafter

TranscriptionInitiation

TrpoperatorRibosomalproteins

TheCaseofPhageλ:LayersofRegulationAminoacidbiosyntheticoperonsarecontrolledbyprematuretranscriptiontermination:thetryptophanoperonThetrp

operonencodesfivestructuralgenesrequiredfortryptophansynthesis.Thesegenesareregulatedtoefficientlyexpressonlywhentryptophanislimiting.Twolayersofregulationareinvolved:

(1)transcriptionrepressionbytheTrprepressor(initiation);(2)attenuationRibosomalproteinsaretranslationalrepressorsoftheirownsynthesisChallenges

theribosomeproteinsynthesis:Eachribosomecontainssome50distinctproteinsthatmustbemadeatthesamerateTherateoftheribosomeproteinsynthesisistightlyclosedtothecell’sgrowthrate

RibosomalproteinoperonsTheproteinthatactsasatranslationalrepressoroftheotherproteinsisshadedred.

Themechanismofoneribosomalproteinalsofunctionsasaregulatorofitsowntranslation:theproteinbindstothesimilarsitesontheribosomalRNAandtotheregulated

mRNAFig16-23PrinciplesofTranscriptionalRegulationRegulationofTranscriptionInitiation:

ExamplesfromBacteria:Lac

operonalternativesfactors,

NtrC,MerR,Galrep

araBAD

operonExamplesofGeneRegulationafterTranscriptionInitiationTheCaseofPhageλ:LayersofRegulationBacteriophage

λisavirusthatinfectsE.coli.Uponinfection,thephagecanpropagateineitheroftwoways:lyticallyorlysogenically.

λhasa50-kbgenomeandsome50genes.Mostoftheseencodecoatproteins,proteinsinvolvedinDNAreplication,recombinationandlysis.AlternativepatternsofgeneexpressioncontrollyticandlysogenicgrowthPromotersintherightandleftcontrolregionsofphageλThedepictedregioncontainstwogenes(cIandcro)andthreepromoters.(PR,PL,PRM)TranscriptionintheλcontrolregionsinlyticandlysogenicgrowthRegulatoryproteinsandtheirbindingsitesThecIencodesλrepressor,aproteinoftwodomainsjoinedbyaflexiblelinkerregion.RelativepositionsofpromoterandoperatorsitesinORλrepressorCroλRepressorBindstoOperatorSitesCooperativelyTheλrepressormonomersinteracttoformdimers,andthosedimersinteracttoformtetramers.TheseinteractionensurethatbindingofrepressortoDNAiscooperative.TheactionofλrepressorandCroRepressorboundtoOR1andOR2turnsofftranscriptionfromPR.RepressorboundatOR2contactsRNApolymeraseat

PRMactivatingexpressionofthecIgene.OR3lieswithinPRM;CroboundthererepressestranscriptionofcI.ResspressorandCrobindinDifferentPatternstoControlLyticandLysogenicGrowthForlyticgrowth,asingleCro

dimerisboundtoOR3;thissiteoverlapsPRMandsoCrorepressesthatpromoter.PRbindsRNApolymeraseanddirectstranscriptionoflyticgenes;PLdoeslikewise.Duringlysogeny,PRMison,whilePRandPLareoff.repressorboundcooperativelyatOR1andOR2blocksRNApolymerasebindingatPRrepressingtranscriptionfromthatpromoter.HowthelysogenicstateswitchestothealternativelyticoneLysogenicinductionrequiresproteolyticcleavageofλrepressorE.colisensesandrespondstoDNAdamage.ItdoesthisbyactivatingthefunctionofaproteincalledRecA.ActivatedRecAstimulatesautocleavageofLexA,releasingrepressionofthosegenes.ThisiscalledtheSOSresponse.Ifthecellisalysogen,λrepressorhasevolvedtoresembleLexA,ensuringthatλrepressortooundergoesautocleavageinresponsetoactivatedRecA.Thelevelofrepressorinalysogenmustbetightlyregulated.Repressorensuresitslevelneverdropstoolow:itactivatesitsownexpression,anexampleofpositiveautoregulation.Repressorensureitslevelnevergettoohigh,repressoralsoregulatesitselfnegatively.(negativeautoregulation)NegativeAutoregulationofRepressorRequiresLong-DistanceInteractionsandaLargeDNALoop

interactionofrepressorsatORandOLAnotherActivator,λcⅡ,

ControlstheDecisionbetweenLyticandLysogenicGrowthuponInfectionofaNewHost.

DeterminewhichwaythephagechooseinthefirstplaceGenesandpromptersinvolvedinthelytic/lysogenicchoiceGrowthConditionsofE.colicontroltheStabilityofCⅡproteinandLytic/LysogenicChoice

EstablishmentoflysogenyCII:unstableproteinFstH:

hflgeneencoding,proteaseregulatedbygrowthconditionofbacteria.

CIIpromoterPI:recombinationCIIpromoterPAQ:promotethelysogenicdevelopmentdegrad