Lac operon - Wikipedia

The lactose operon (lac operon) is an operon required for the transport and metabolism of lactose in E. coli and many other enteric bacteria. lacoperon FromWikipedia,thefreeencyclopedia Jumptonavigation Jumptosearch Setgenesencodingproteinsandenzymesforlactosemetabolism Thelactoseoperon(lacoperon)isanoperonrequiredforthetransportandmetabolismoflactoseinE.coliandmanyotherentericbacteria.Althoughglucoseisthepreferredcarbonsourceformostbacteria,thelacoperonallowsfortheeffectivedigestionoflactosewhenglucoseisnotavailablethroughtheactivityofbeta-galactosidase.[1]Generegulationofthelacoperonwasthefirstgeneticregulatorymechanismtobeunderstoodclearly,soithasbecomeaforemostexampleofprokaryoticgeneregulation.Itisoftendiscussedinintroductorymolecularandcellularbiologyclassesforthisreason.ThislactosemetabolismsystemwasusedbyFrançoisJacobandJacquesMonodtodeterminehowabiologicalcellknowswhichenzymetosynthesize.TheirworkonthelacoperonwonthemtheNobelPrizeinPhysiologyin1965.[1] BacterialoperonsarepolycistronictranscriptsthatareabletoproducemultipleproteinsfromonemRNAtranscript.Inthiscase,whenlactoseisrequiredasasugarsourceforthebacterium,thethreegenesofthelacoperoncanbeexpressedandtheirsubsequentproteinstranslated:lacZ,lacY,andlacA.ThegeneproductoflacZisβ-galactosidasewhichcleaveslactose,adisaccharide,intoglucoseandgalactose.lacYencodesBeta-galactosidepermease,amembraneproteinwhichbecomesembeddedinthecytoplasmicmembranetoenablethecellulartransportoflactoseintothecell.Finally,lacAencodesGalactosideacetyltransferase. Thelacoperon.Top:Repressed,Bottom:Active.1:RNApolymerase,2:Repressor,3:Promoter,4:Operator,5:Lactose,6:lacZ,7:lacY,8:lacA. Itwouldbewastefultoproduceenzymeswhennolactoseisavailableorifapreferableenergysourcesuchasglucosewereavailable.Thelacoperonusesatwo-partcontrolmechanismtoensurethatthecellexpendsenergyproducingtheenzymesencodedbythelacoperononlywhennecessary.[2]Intheabsenceoflactose,thelacrepressor,lacI,haltsproductionoftheenzymesencodedbythelacoperon.[3]Thelacrepressorisalwaysexpressed,unlessaco-inducerbindstoit.Inotherwords,itistranscribedonlyinthepresenceofsmallco-inducermolecules.Inthepresenceofglucose,thecataboliteactivatorprotein(CAP),requiredforproductionoftheenzymes,remainsinactive,andEIIAGlcshutsdownlactosepermeasetopreventtransportoflactoseintothecell.Thisdualcontrolmechanismcausesthesequentialutilizationofglucoseandlactoseintwodistinctgrowthphases,knownasdiauxie. Contents 1Structure 1.1Geneticnomenclature 2Regulation 2.1Repressorstructure 2.1.1Mechanismofinduction 2.1.2Roleofnon-specificbinding 2.2Lactoseanalogs 3Developmentoftheclassicmodel 3.1Classificationofregulatorymutants 3.2RegulationbycyclicAMP[19] 4Useinmolecularbiology 5Seealso 6References 7Externallinks Structure[edit] Structureoflactoseandtheproductsofitscleavage. Thelacoperonconsistsof3structuralgenes,andapromoter,aterminator,regulator,andanoperator.Thethreestructuralgenesare:lacZ,lacY,andlacA. lacZencodesβ-galactosidase(LacZ),anintracellularenzymethatcleavesthedisaccharidelactoseintoglucoseandgalactose. lacYencodesBeta-galactosidepermease(LacY),atransmembranesymporterthatpumpsβ-galactosidesincludinglactoseintothecellusingaprotongradientinthesamedirection.Permeaseincreasesthepermeabilityofthecelltoβ-galactosides. lacAencodesβ-galactosidetransacetylase(LacA),anenzymethattransfersanacetylgroupfromacetyl-CoAtothiogalactoside. OnlylacZandlacYappeartobenecessaryforlactosecatabolicpathway. Geneticnomenclature[edit] Three-letterabbreviationsareusedtodescribephenotypesinbacteriaincludingE.coli. Examplesinclude: Lac(theabilitytouselactose), His(theabilitytosynthesizetheaminoacidhistidine) Mot(swimmingmotility) SmR(resistancetotheantibioticstreptomycin) InthecaseofLac,wildtypecellsareLac+andareabletouselactoseasacarbonandenergysource,whileLac−mutantderivativescannotuselactose.Thesamethreelettersaretypicallyused(lower-case,italicized)tolabelthegenesinvolvedinaparticularphenotype,whereeachdifferentgeneisadditionallydistinguishedbyanextraletter.ThelacgenesencodingenzymesarelacZ,lacY,andlacA.ThefourthlacgeneislacI,encodingthelactoserepressor—"I"standsforinducibility. Onemaydistinguishbetweenstructuralgenesencodingenzymes,andregulatorygenesencodingproteinsthataffectgeneexpression.Currentusageexpandsthephenotypicnomenclaturetoapplytoproteins:thus,LacZistheproteinproductofthelacZgene,β-galactosidase.Variousshortsequencesthatarenotgenesalsoaffectgeneexpression,includingthelacpromoter,lacp,andthelacoperator,laco.Althoughitisnotstrictlystandardusage,mutationsaffectinglacoarereferredtoaslacoc,forhistoricalreasons. Inducer Regulation[edit] Specificcontrolofthelacgenesdependsontheavailabilityofthesubstratelactosetothebacterium.Theproteinsarenotproducedbythebacteriumwhenlactoseisunavailableasacarbonsource. Thelacgenesareorganizedintoanoperon;thatis,theyareorientedinthesamedirectionimmediatelyadjacentonthechromosomeandareco-transcribedintoasinglepolycistronicmRNAmolecule.TranscriptionofallgenesstartswiththebindingoftheenzymeRNApolymerase(RNAP),aDNA-bindingprotein,whichbindstoaspecificDNAbindingsite,thepromoter,immediatelyupstreamofthegenes.BindingofRNApolymerasetothepromoterisaidedbythecAMP-boundcataboliteactivatorprotein(CAP,alsoknownasthecAMPreceptorprotein).[4]However,thelacIgene(regulatorygeneforlacoperon)producesaproteinthatblocksRNAPfrombindingtotheoperatoroftheoperon.Thisproteincanonlyberemovedwhenallolactosebindstoit,andinactivatesit.TheproteinthatisformedbythelacIgeneisknownasthelacrepressor.Thetypeofregulationthatthelacoperonundergoesisreferredtoasnegativeinducible,meaningthatthegeneisturnedoffbytheregulatoryfactor(lacrepressor)unlesssomemolecule(lactose)isadded.Becauseofthepresenceofthelacrepressorprotein,geneticengineerswhoreplacethelacZgenewithanothergenewillhavetogrowtheexperimentalbacteriaonagarwithlactoseavailableonit.Iftheydonot,thegenetheyaretryingtoexpresswillnotbeexpressedastherepressorproteinisstillblockingRNAPfrombindingtothepromoterandtranscribingthegene.Oncetherepressorisremoved,RNAPthenproceedstotranscribeallthreegenes(lacZYA)intomRNA.EachofthethreegenesonthemRNAstrandhasitsownShine-Dalgarnosequence,sothegenesareindependentlytranslated.[5]TheDNAsequenceoftheE.colilacoperon,thelacZYAmRNA,andthelacIgenesareavailablefromGenBank(view). Thefirstcontrolmechanismistheregulatoryresponsetolactose,whichusesanintracellularregulatoryproteincalledthelactoserepressortohinderproductionofβ-galactosidaseintheabsenceoflactose.ThelacIgenecodingfortherepressorliesnearbythelacoperonandisalwaysexpressed(constitutive).Iflactoseismissingfromthegrowthmedium,therepressorbindsverytightlytoashortDNAsequencejustdownstreamofthepromoternearthebeginningoflacZcalledthelacoperator.TherepressorbindingtotheoperatorinterfereswithbindingofRNAPtothepromoter,andthereforemRNAencodingLacZandLacYisonlymadeatverylowlevels.Whencellsaregrowninthepresenceoflactose,however,alactosemetabolitecalledallolactose,madefromlactosebytheproductofthelacZgene,bindstotherepressor,causinganallostericshift.Thusaltered,therepressorisunabletobindtotheoperator,allowingRNAPtotranscribethelacgenesandtherebyleadingtohigherlevelsoftheencodedproteins. Thesecondcontrolmechanismisaresponsetoglucose,whichusesthecataboliteactivatorprotein(CAP)homodimertogreatlyincreaseproductionofβ-galactosidaseintheabsenceofglucose.Cyclicadenosinemonophosphate(cAMP)isasignalmoleculewhoseprevalenceisinverselyproportionaltothatofglucose.ItbindstotheCAP,whichinturnallowstheCAPtobindtotheCAPbindingsite(a16bpDNAsequenceupstreamofthepromoterontheleftinthediagrambelow,about60bpupstreamofthetranscriptionstartsite),[6]whichassiststheRNAPinbindingtotheDNA.Intheabsenceofglucose,thecAMPconcentrationishighandbindingofCAP-cAMPtotheDNAsignificantlyincreasestheproductionofβ-galactosidase,enablingthecelltohydrolyselactoseandreleasegalactoseandglucose. Morerecentlyinducerexclusionwasshowntoblockexpressionofthelacoperonwhenglucoseispresent.GlucoseistransportedintothecellbythePEP-dependentphosphotransferasesystem.ThephosphategroupofphosphoenolpyruvateistransferredviaaphosphorylationcascadeconsistingofthegeneralPTS(phosphotransferasesystem)proteinsHPrandEIAandtheglucose-specificPTSproteinsEIIAGlcandEIIBGlc,thecytoplasmicdomainoftheEIIglucosetransporter.TransportofglucoseisaccompaniedbyitsphosphorylationbyEIIBGlc,drainingthephosphategroupfromtheotherPTSproteins,includingEIIAGlc.TheunphosphorylatedformofEIIAGlcbindstothelacpermeaseandpreventsitfrombringinglactoseintothecell.Therefore,ifbothglucoseandlactosearepresent,thetransportofglucoseblocksthetransportoftheinducerofthelacoperon.[7] Repressorstructure[edit] TetramericLacIbindstwooperatorsequencesandinducesDNAlooping.TwodimericLacIfunctionalsubunits(red+blueandgreen+orange)eachbindaDNAoperatorsequence(labeled).Thesetwofunctionalsubunitsarecoupledatthetetramerizationregion(labeled);thus,tetramericLacIbindstwooperatorsequences.ThisallowstetramericLacItoinduceDNAlooping. Thelacrepressorisafour-partprotein,atetramer,withidenticalsubunits.Eachsubunitcontainsahelix-turn-helix(HTH)motifcapableofbindingtoDNA.TheoperatorsitewhererepressorbindsisaDNAsequencewithinvertedrepeatsymmetry.ThetwoDNAhalf-sitesoftheoperatortogetherbindtotwoofthesubunitsoftherepressor.Althoughtheothertwosubunitsofrepressorarenotdoinganythinginthismodel,thispropertywasnotunderstoodformanyyears. Eventuallyitwasdiscoveredthattwoadditionaloperatorsareinvolvedinlacregulation.[8]One(O3)liesabout−90bpupstreamofO1intheendofthelacIgene,andtheother(O2)isabout+410bpdownstreamofO1intheearlypartoflacZ.Thesetwositeswerenotfoundintheearlyworkbecausetheyhaveredundantfunctionsandindividualmutationsdonotaffectrepressionverymuch.SinglemutationstoeitherO2orO3haveonly2to3-foldeffects.However,theirimportanceisdemonstratedbythefactthatadoublemutantdefectiveinbothO2andO3isdramaticallyde-repressed(byabout70-fold). Inthecurrentmodel,lacrepressorisboundsimultaneouslytoboththemainoperatorO1andtoeitherO2orO3.TheinterveningDNAloopsoutfromthecomplex.Theredundantnatureofthetwominoroperatorssuggeststhatitisnotaspecificloopedcomplexthatisimportant.Oneideaisthatthesystemworksthroughtethering;ifboundrepressorreleasesfromO1momentarily,bindingtoaminoroperatorkeepsitinthevicinity,sothatitmayrebindquickly.ThiswouldincreasetheaffinityofrepressorforO1. Mechanismofinduction[edit] 1:RNAPolymerase,2:Repressor,3:Promoter,4:Operator,5:Lactose,6:lacZ,7:lacY,8:lacA.Top:Thegeneisessentiallyturnedoff.Thereisnoallolactosetoinhibitthelacrepressor,sotherepressorbindstightlytotheoperator,whichobstructstheRNApolymerasefrombindingtothepromoter,resultinginnolaczyamRNAtranscripts.Bottom:Thegeneisturnedon.Allolactoseinhibitstherepressor,allowingtheRNApolymerasetobindtothepromoterandexpressthegenes,resultinginproductionofLacZYA.Eventually,theenzymeswilldigestallofthelactose,untilthereisnoallolactosethatcanbindtotherepressor.Therepressorwillthenbindtotheoperator,stoppingthetranscriptionoftheLacZYAgenes. Therepressorisanallostericprotein,i.e.itcanassumeeitheroneoftwoslightlydifferentshapes,whichareinequilibriumwitheachother.InoneformtherepressorwillbindtotheoperatorDNAwithhighspecificity,andintheotherformithaslostitsspecificity.Accordingtotheclassicalmodelofinduction,bindingoftheinducer,eitherallolactoseorIPTG,totherepressoraffectsthedistributionofrepressorbetweenthetwoshapes.Thus,repressorwithinducerboundisstabilizedinthenon-DNA-bindingconformation.However,thissimplemodelcannotbethewholestory,becauserepressorisboundquitestablytoDNA,yetitisreleasedrapidlybyadditionofinducer.Therefore,itseemsclearthataninducercanalsobindtotherepressorwhentherepressorisalreadyboundtoDNA.Itisstillnotentirelyknownwhattheexactmechanismofbindingis.[9] Roleofnon-specificbinding[edit] Non-specificbindingoftherepressortoDNAplaysacrucialroleintherepressionandinductionoftheLac-operon.ThespecificbindingsitefortheLac-repressorproteinistheoperator.Thenon-specificinteractionismediatedmainlybycharge-chargeinteractionswhilebindingtotheoperatorisreinforcedbyhydrophobicinteractions.Additionally,thereisanabundanceofnon-specificDNAsequencestowhichtherepressorcanbind.Essentially,anysequencethatisnottheoperator,isconsiderednon-specific.Studieshaveshown,thatwithoutthepresenceofnon-specificbinding,induction(orunrepression)oftheLac-operoncouldnotoccurevenwithsaturatedlevelsofinducer.Ithadbeendemonstratedthat,withoutnon-specificbinding,thebasallevelofinductionistenthousandtimessmallerthanobservednormally.Thisisbecausethenon-specificDNAactsassortofa"sink"fortherepressorproteins,distractingthemfromtheoperator.Thenon-specificsequencesdecreasetheamountofavailablerepressorinthecell.Thisinturnreducestheamountofinducerrequiredtounrepressthesystem.[10] Lactoseanalogs[edit] IPTG ONPG X-gal allolactose Anumberoflactosederivativesoranalogshavebeendescribedthatareusefulforworkwiththelacoperon.Thesecompoundsaremainlysubstitutedgalactosides,wheretheglucosemoietyoflactoseisreplacedbyanotherchemicalgroup. Isopropyl-β-D-thiogalactopyranoside(IPTG)isfrequentlyusedasaninducerofthelacoperonforphysiologicalwork.[1]IPTGbindstorepressorandinactivatesit,butisnotasubstrateforβ-galactosidase.OneadvantageofIPTGforinvivostudiesisthatsinceitcannotbemetabolizedbyE.coli.Itsconcentrationremainsconstantandtherateofexpressionoflacp/o-controlledgenesisnotavariableintheexperiment.IPTGintakeisdependentontheactionoflactosepermeaseinP.fluorescens,butnotinE.coli.[11] Phenyl-β-D-galactose(phenyl-Gal)isasubstrateforβ-galactosidase,butdoesnotinactivaterepressorandsoisnotaninducer.Sincewildtypecellsproduceverylittleβ-galactosidase,theycannotgrowonphenyl-Galasacarbonandenergysource.Mutantslackingrepressorareabletogrowonphenyl-Gal.Thus,minimalmediumcontainingonlyphenyl-Galasasourceofcarbonandenergyisselectiveforrepressormutantsandoperatormutants.If108cellsofawildtypestrainareplatedonagarplatescontainingphenyl-Gal,therarecolonieswhichgrowaremainlyspontaneousmutantsaffectingtherepressor.Therelativedistributionofrepressorandoperatormutantsisaffectedbythetargetsize.SincethelacIgeneencodingrepressorisabout50timeslargerthantheoperator,repressormutantspredominateintheselection. Thiomethylgalactoside[TMG]isanotherlactoseanalog.TheseinhibitthelacIrepressor.Atlowinducerconcentrations,bothTMGandIPTGcanenterthecellthroughthelactosepermease.Howeverathighinducerconcentrations,bothanalogscanenterthecellindependently.TMGcanreducegrowthratesathighextracellularconcentrations.[12] Othercompoundsserveascolorfulindicatorsofβ-galactosidaseactivity. ONPGiscleavedtoproducetheintenselyyellowcompound,orthonitrophenolandgalactose,andiscommonlyusedasasubstrateforassayofβ-galactosidaseinvitro.[13] Coloniesthatproduceβ-galactosidaseareturnedbluebyX-gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside)whichisanartificialsubstrateforB-galactosidasewhosecleavageresultsingalactoseand4-Cl,3-Brindigothusproducingadeepbluecolor.[14] Allolactoseisanisomeroflactoseandistheinducerofthelacoperon.Lactoseisgalactose-β(1→4)-glucose,whereasallolactoseisgalactose-β(1→6)-glucose.Lactoseisconvertedtoallolactosebyβ-galactosidaseinanalternativereactiontothehydrolyticone.AphysiologicalexperimentwhichdemonstratestheroleofLacZinproductionofthe"true"inducerinE.colicellsistheobservationthatanullmutantoflacZcanstillproduceLacYpermeasewhengrownwithIPTG,anon-hydrolyzableanalogofallolactose,butnotwhengrownwithlactose.Theexplanationisthatprocessingoflactosetoallolactose(catalyzedbyβ-galactosidase)isneededtoproducetheinducerinsidethecell. Developmentoftheclassicmodel[edit] TheexperimentalmicroorganismusedbyFrançoisJacobandJacquesMonodwasthecommonlaboratorybacterium,E.coli,butmanyofthebasicregulatoryconceptsthatwerediscoveredbyJacobandMonodarefundamentaltocellularregulationinallorganisms.[15]Thekeyideaisthatproteinsarenotsynthesizedwhentheyarenotneeded—E.coliconservescellularresourcesandenergybynotmakingthethreeLacproteinswhenthereisnoneedtometabolizelactose,suchaswhenothersugarslikeglucoseareavailable.ThefollowingsectiondiscusseshowE.colicontrolscertaingenesinresponsetometabolicneeds. DuringWorldWarII,MonodwastestingtheeffectsofcombinationsofsugarsasnutrientsourcesforE.coliandB.subtilis.Monodwasfollowinguponsimilarstudiesthathadbeenconductedbyotherscientistswithbacteriaandyeast.Hefoundthatbacteriagrownwithtwodifferentsugarsoftendisplayedtwophasesofgrowth.Forexample,ifglucoseandlactosewerebothprovided,glucosewasmetabolizedfirst(growthphaseI,seeFigure2)andthenlactose(growthphaseII).Lactosewasnotmetabolizedduringthefirstpartofthediauxicgrowthcurvebecauseβ-galactosidasewasnotmadewhenbothglucoseandlactosewerepresentinthemedium.Monodnamedthisphenomenondiauxie.[16] Figure2:Monod's"bi-phasic"growthcurve Monodthenfocusedhisattentionontheinductionofβ-galactosidaseformationthatoccurredwhenlactosewasthesolesugarintheculturemedium.[17] Classificationofregulatorymutants[edit] AconceptualbreakthroughofJacobandMonod[18]wastorecognizethedistinctionbetweenregulatorysubstancesandsiteswheretheyacttochangegeneexpression.Aformersoldier,Jacobusedtheanalogyofabomberthatwouldreleaseitslethalcargouponreceiptofaspecialradiotransmissionorsignal.Aworkingsystemrequiresbothagroundtransmitterandareceiverintheairplane.Now,supposethattheusualtransmitterisbroken.Thissystemcanbemadetoworkbyintroductionofasecond,functionaltransmitter.Incontrast,hesaid,considerabomberwithadefectivereceiver.Thebehaviorofthisbombercannotbechangedbyintroductionofasecond,functionalaeroplane. Toanalyzeregulatorymutantsofthelacoperon,Jacobdevelopedasystembywhichasecondcopyofthelacgenes(lacIwithitspromoter,andlacZYAwithpromoterandoperator)couldbeintroducedintoasinglecell.Acultureofsuchbacteria,whicharediploidforthelacgenesbutotherwisenormal,isthentestedfortheregulatoryphenotype.Inparticular,itisdeterminedwhetherLacZandLacYaremadeevenintheabsenceofIPTG(duetothelactoserepressorproducedbythemutantgenebeingnon-functional).Thisexperiment,inwhichgenesorgeneclustersaretestedpairwise,iscalledacomplementationtest. Thistestisillustratedinthefigure(lacAisomittedforsimplicity).First,certainhaploidstatesareshown(i.e.thecellcarriesonlyasinglecopyofthelacgenes).Panel(a)showsrepression,(b)showsinductionbyIPTG,and(c)and(d)showtheeffectofamutationtothelacIgeneortotheoperator,respectively.Inpanel(e)thecomplementationtestforrepressorisshown.IfonecopyofthelacgenescarriesamutationinlacI,butthesecondcopyiswildtypeforlacI,theresultingphenotypeisnormal—butlacZisexpressedwhenexposedtoinducerIPTG.Mutationsaffectingrepressoraresaidtoberecessivetowildtype(andthatwildtypeisdominant),andthisisexplainedbythefactthatrepressorisasmallproteinwhichcandiffuseinthecell.ThecopyofthelacoperonadjacenttothedefectivelacIgeneiseffectivelyshutoffbyproteinproducedfromthesecondcopyoflacI. Ifthesameexperimentiscarriedoutusinganoperatormutation,adifferentresultisobtained(panel(f)).ThephenotypeofacellcarryingonemutantandonewildtypeoperatorsiteisthatLacZandLacYareproducedevenintheabsenceoftheinducerIPTG;becausethedamagedoperatorsite,doesnotpermitbindingoftherepressortoinhibittranscriptionofthestructuralgenes.Theoperatormutationisdominant.Whentheoperatorsitewhererepressormustbindisdamagedbymutation,thepresenceofasecondfunctionalsiteinthesamecellmakesnodifferencetoexpressionofgenescontrolledbythemutantsite. Amoresophisticatedversionofthisexperimentusesmarkedoperonstodistinguishbetweenthetwocopiesofthelacgenesandshowthattheunregulatedstructuralgene(s)is(are)theone(s)nexttothemutantoperator(panel(g).Forexample,supposethatonecopyismarkedbyamutationinactivatinglacZsothatitcanonlyproducetheLacYprotein,whilethesecondcopycarriesamutationaffectinglacYandcanonlyproduceLacZ.Inthisversion,onlythecopyofthelacoperonthatisadjacenttothemutantoperatorisexpressedwithoutIPTG.Wesaythattheoperatormutationiscis-dominant,itisdominanttowildtypebutaffectsonlythecopyoftheoperonwhichisimmediatelyadjacenttoit. Thisexplanationismisleadinginanimportantsense,becauseitproceedsfromadescriptionoftheexperimentandthenexplainstheresultsintermsofamodel.Butinfact,itisoftentruethatthemodelcomesfirst,andanexperimentisfashionedspecificallytotestthemodel.JacobandMonodfirstimaginedthattheremustbeasiteinDNAwiththepropertiesoftheoperator,andthendesignedtheircomplementationteststoshowthis. Thedominanceofoperatormutantsalsosuggestsaproceduretoselectthemspecifically.Ifregulatorymutantsareselectedfromacultureofwildtypeusingphenyl-Gal,asdescribedabove,operatormutationsarerarecomparedtorepressormutantsbecausethetarget-sizeissosmall.Butifinsteadwestartwithastrainwhichcarriestwocopiesofthewholelacregion(thatisdiploidforlac),therepressormutations(whichstilloccur)arenotrecoveredbecausecomplementationbythesecond,wildtypelacIgeneconfersawildtypephenotype.Incontrast,mutationofonecopyoftheoperatorconfersamutantphenotypebecauseitisdominanttothesecond,wildtypecopy. RegulationbycyclicAMP[19][edit] Explanationofdiauxiedependedonthecharacterizationofadditionalmutationsaffectingthelacgenesotherthanthoseexplainedbytheclassicalmodel.Twoothergenes,cyaandcrp,subsequentlywereidentifiedthatmappedfarfromlac,andthat,whenmutated,resultinadecreasedlevelofexpressioninthepresenceofIPTGandeveninstrainsofthebacteriumlackingtherepressororoperator.ThediscoveryofcAMPinE.coliledtothedemonstrationthatmutantsdefectivethecyagenebutnotthecrpgenecouldberestoredtofullactivitybytheadditionofcAMPtothemedium. Thecyageneencodesadenylatecyclase,whichproducescAMP.Inacyamutant,theabsenceofcAMPmakestheexpressionofthelacZYAgenesmorethantentimeslowerthannormal.AdditionofcAMPcorrectsthelowLacexpressioncharacteristicofcyamutants.Thesecondgene,crp,encodesaproteincalledcataboliteactivatorprotein(CAP)orcAMPreceptorprotein(CRP).[20] Howeverthelactosemetabolismenzymesaremadeinsmallquantitiesinthepresenceofbothglucoseandlactose(sometimescalledleakyexpression)duetothefactthattheLacIrepressorrapidlyassociates/dissociatesfromtheDNAratherthantightlybindingtoit,whichcanallowtimeforRNAPtobindandtranscribemRNAsoflacZYA.Leakyexpressionisnecessaryinordertoallowformetabolismofsomelactoseaftertheglucosesourceisexpended,butbeforelacexpressionisfullyactivated. Insummary: WhenlactoseisabsentthenthereisverylittleLacenzymeproduction(theoperatorhasLacrepressorboundtoit). Whenlactoseispresentbutapreferredcarbonsource(likeglucose)isalsopresentthenasmallamountofenzymeisproduced(Lacrepressorisnotboundtotheoperator). Whenglucoseisabsent,CAP-cAMPbindstoaspecificDNAsiteupstreamofthepromoterandmakesadirectprotein-proteininteractionwithRNAPthatfacilitatesthebindingofRNAPtothepromoter. Thedelaybetweengrowthphasesreflectsthetimeneededtoproducesufficientquantitiesoflactose-metabolizingenzymes.First,theCAPregulatoryproteinhastoassembleonthelacpromoter,resultinginanincreaseintheproductionoflacmRNA.MoreavailablecopiesofthelacmRNAresultsintheproduction(seetranslation)ofsignificantlymorecopiesofLacZ(β-galactosidase,forlactosemetabolism)andLacY(lactosepermeasetotransportlactoseintothecell).Afteradelayneededtoincreasethelevelofthelactosemetabolizingenzymes,thebacteriaenterintoanewrapidphaseofcellgrowth. lacoperonindetail TwopuzzlesofcataboliterepressionrelatetohowcAMPlevelsarecoupledtothepresenceofglucose,andsecondly,whythecellsshouldevenbother.Afterlactoseiscleaveditactuallyformsglucoseandgalactose(easilyconvertedtoglucose).Inmetabolicterms,lactoseisjustasgoodacarbonandenergysourceasglucose.ThecAMPlevelisrelatednottointracellularglucoseconcentrationbuttotherateofglucosetransport,whichinfluencestheactivityofadenylatecyclase.(Inaddition,glucosetransportalsoleadstodirectinhibitionofthelactosepermease.)AstowhyE.coliworksthisway,onecanonlyspeculate.Allentericbacteriafermentglucose,whichsuggeststheyencounteritfrequently.Itispossiblethatasmalldifferenceinefficiencyoftransportormetabolismofglucosev.lactosemakesitadvantageousforcellstoregulatethelacoperoninthisway.[21] Useinmolecularbiology[edit] Thelacgeneanditsderivativesareamenabletouseasareportergeneinanumberofbacterial-basedselectiontechniquessuchastwohybridanalysis,inwhichthesuccessfulbindingofatranscriptionalactivatortoaspecificpromotersequencemustbedetermined.[14]InLBplatescontainingX-gal,thecolourchangefromwhitecoloniestoashadeofbluecorrespondstoabout20–100β-galactosidaseunits,whiletetrazoliumlactoseandMacConkeylactosemediahavearangeof100–1000units,beingmostsensitiveinthehighandlowpartsofthisrangerespectively.[14]SinceMacConkeylactoseandtetrazoliumlactosemediabothrelyontheproductsoflactosebreakdown,theyrequirethepresenceofbothlacZandlacYgenes.ThemanylacfusiontechniqueswhichincludeonlythelacZgenearethussuitedtoX-galplates[14]orONPGliquidbroths.[22] Seealso[edit] Cataboliterepression References[edit] ^abGriffiths,AnthonyJ.F.;Wessler,SusanR.;Carroll,SeanB.;Doebley,John(2015).AnIntroductiontoGeneticAnalysis(11 ed.).Freeman,W.H.&Company.pp. 400–412.ISBN 9781464109485. ^McClean,Phillip(1997)."ProkaryoticGeneExpression".ndsu.edu.Retrieved19May2017. ^"ProkaryoticGeneExpression".ndsu.edu.Retrieved19May2017. ^BusbyS.,EbrightRH.(2001)."Transcriptionactivationbycataboliteactivatorprotein(CAP)".J.Mol.Biol.293(2):199–213.doi:10.1006/jmbi.1999.3161.PMID 10550204. ^Kennell,David;Riezman,Howard(July1977)."TranscriptionandtranslationinitiationfrequenciesoftheEscherichiacolilacoperon".JournalofMolecularBiology.114(1):1–21.doi:10.1016/0022-2836(77)90279-0.PMID 409848. ^Malan,T.Philip;Kolb,Annie;Buc,Henri;McClure,William(December1984)."MechanismofCRP-cAMPActivationoflacOperonTranscriptionInitiationActivationoftheP1Promoter".J.Mol.Biol.180(4):881–909.doi:10.1016/0022-2836(84)90262-6.PMID 6098691. ^GörkeB,StülkeJ(August2008)."Carboncataboliterepressioninbacteria:manywaystomakethemostoutofnutrients".NatureReviews.Microbiology.6(8):613–24.doi:10.1038/nrmicro1932.PMID 18628769.S2CID 8782171. ^Oehler,S.;Eismann,E.R.;Krämer,H.;Müller-Hill,B.(1990)."Thethreeoperatorsofthelacoperoncooperateinrepression".TheEMBOJournal.9(4):973–979.doi:10.1002/j.1460-2075.1990.tb08199.x.PMC 551766.PMID 2182324. ^Griffiths,AnthonyJF;Gelbart,WilliamM.;Miller,JeffreyH.;Lewontin,RichardC.(1999)."RegulationoftheLactoseSystem".ModernGeneticAnalysis.NewYork:W.H.Freeman.ISBN 0-7167-3118-5. ^vonHippel,P.H.;Revzin,A.;Gross,C.A.;Wang,A.C.(December1974)."Non-specificDNAbindingofgenomeregulatingproteinsasabiologicalcontrolmechanism:I.Thelacoperon:equilibriumaspects".PNAS.71(12):4808–12.doi:10.1073/pnas.71.12.4808.PMC 433986.PMID 4612528. ^HansenLH,KnudsenS,SørensenSJ(June1998)."TheeffectofthelacYgeneontheinductionofIPTGinduciblepromoters,studiedinEscherichiacoliandPseudomonasfluorescens".Curr.Microbiol.36(6):341–7.doi:10.1007/s002849900320.PMID 9608745.S2CID 22257399.Archivedfromtheoriginalon18October2000. ^MarbachA,BettenbrockK(January2012)."lacoperoninductioninEscherichiacoli:SystematiccomparisonofIPTGandTMGinductionandinfluenceofthetransacetylaseLacA".JournalofBiotechnology.157(1):82–88.doi:10.1016/j.jbiotec.2011.10.009.PMID 22079752. ^"ONPG(β-Galactosidase)test".September2000.Archivedfromtheoriginalon3November2007.Retrieved25October2007. ^abcdJoungJ,RammE,PaboC(2000)."Abacterialtwo-hybridselectionsystemforstudyingprotein–DNAandprotein–proteininteractions".ProcNatlAcadSciUSA.97(13):7382–7.Bibcode:2000PNAS...97.7382J.doi:10.1073/pnas.110149297.PMC 16554.PMID 10852947. ^"Milestone2–Avisionarypair :NatureMilestonesingeneexpression".www.nature.com.Retrieved27December2015. ^Muller-Hill,Benno(1996).ThelacOperon,aShortHistoryofaGeneticParadigm.Berlin:WalterdeGruyter.pp. 7–10.ISBN 3-11-014830-7. ^McKnight,StevenL.(1992).TranscriptionalRegulation.ColdSpringHarbor,NY:ColdSpringHarborLaboratoryPress.pp. 3–24.ISBN 0-87969-410-6. ^JacobF.;MonodJ(June1961)."Geneticregulatorymechanismsinthesynthesisofproteins".JMolBiol.3(3):318–56.doi:10.1016/S0022-2836(61)80072-7.PMID 13718526. ^Montminy,M.(1997)."TranscriptionalregulationbycyclicAMP".AnnualReviewofBiochemistry.66:807–822.doi:10.1146/annurev.biochem.66.1.807.ISSN 0066-4154.PMID 9242925. ^Botsford,JL;Harman,JG(March1992)."CyclicAMPinprokaryotes".MicrobiologicalReviews.56(1):100–122.doi:10.1128/MMBR.56.1.100-122.1992.ISSN 0146-0749.PMC 372856.PMID 1315922. ^VazquezA,BegQK,DemenezesMA,et al.(2008)."ImpactofthesolventcapacityconstraintonE.colimetabolism".BMCSystBiol.2:7.doi:10.1186/1752-0509-2-7.PMC 2270259.PMID 18215292. ^"InductionofthelacoperoninE.coli"(PDF).SAPS.Retrieved29June2016. Externallinks[edit] Lac+OperonattheUSNationalLibraryofMedicineMedicalSubjectHeadings(MeSH) lacoperoninNCBIBookshelf[2] VirtualCellAnimationCollectionIntroducing:TheLacOperon ThelacOperon:BozemanScience StainingWholeMouseEmbryosforβ-Galactosidase(lacZ)Activity vteTranscription(Bacterial,Eukaryotic)Transcriptionalregulationprokaryotic Operon lacoperon trpoperon gaboperon araoperon galoperon Repressor lacrepressor trprepressor eukaryoticHistone-modifyingenzymes(histone/nucleosome): Histonemethylation/Histonemethyltransferase EZH2 Histonedemethylase Histoneacetylationanddeacetylation HistonedeacetylaseHDAC1 Histoneacetyltransferase DNAmethylation: DNAmethyltransferase Chromatinremodeling: CHD7 both Transcriptioncoregulator Coactivator Corepressor Inducer Promotion Promoter Pribnowbox TATAbox BRE CAATbox Responseelement Enhancer E-box Responseelement Insulator Silencer Internalcontrolregion Initiation Bacterial Eukaryotic ArchaealtranscriptionfactorB Elongation bacterialRNApolymerase:rpoB eukaryoticRNApolymerase:RNApolymeraseII Termination(bacterial,eukaryotic) Terminator Intrinsictermination Rhofactor vteThedevelopmentofphenotypeKeyconcepts Genotype–phenotypedistinction Reactionnorm Gene–environmentinteraction Gene–environmentcorrelation Operon Heritability Quantitativegenetics Heterochrony Neoteny Heterotopy Geneticarchitecture Canalisation Geneticassimilation Dominance Epistasis Fitnesslandscape/evolutionarylandscape Pleiotropy Plasticity Polygenicinheritance Transgressivesegregation Sequencespace Non-geneticinfluences Epigenetics Maternaleffect Genomicimprinting Dualinheritancetheory Polyphenism Developmentalarchitecture Developmentalbiology Morphogenesis Eyespot Patternformation Segmentation Metamerism Modularity Evolutionofgeneticsystems Evolvability Robustness Neutralnetworks Evolutionofsexualreproduction ControlofdevelopmentSystems Regulationofgeneexpression Generegulatorynetwork Evo-devogenetoolkit Evolutionarydevelopmentalbiology Homeobox Hedgehogsignalingpathway Notchsignalingpathway Elements Homeoticgene Hoxgene Paxgenes eyelessgene Distal-less Engrailed cis-regulatoryelement Ligand Morphogen Cellsurfacereceptor Transcriptionfactor Influentialfigures C.H.Waddington RichardLewontin FrançoisJacob+JacquesMonod Lacoperon EricF.Wieschaus ChristianeNüsslein-Volhard WilliamMcGinnis MikeLevine SeanB.Carroll EndlessFormsMostBeautiful Debates Natureversusnurture Morphogeneticfield Indexofevolutionarybiologyarticles Portal: Biology Retrievedfrom"https://en.wikipedia.org/w/index.php?title=Lac_operon&oldid=1057714418" Categories:GeneexpressionBacterialgeneticsOperonsHiddencategories:ArticleswithshortdescriptionShortdescriptionmatchesWikidataUsedmydatesfromJune2019 Navigationmenu Personaltools NotloggedinTalkContributionsCreateaccountLogin Namespaces ArticleTalk English Views ReadEditViewhistory More Search Navigation MainpageContentsCurrenteventsRandomarticleAboutWikipediaContactusDonate Contribute HelpLearntoeditCommunityportalRecentchangesUploadfile Tools WhatlinkshereRelatedchangesUploadfileSpecialpagesPermanentlinkPageinformationCitethispageWikidataitem Print/export DownloadasPDFPrintableversion Inotherprojects WikimediaCommons Languages CatalàČeštinaDeutschEestiEspañolEuskaraفارسیFrançaisGalego한국어BahasaIndonesiaעבריתಕನ್ನಡNederlands日本語OccitanPolskiPortuguêsРусскийSlovenčinaSlovenščinaSvenskaไทยTiếngViệt中文 Editlinks