PAGE

AnswerstoWeaverendofchapterquestions

Chapter7Operons:FineControlofProkaryoticTranscription

SeeFigure7.1.Duringtheinitialphaseofgrowth,thebacteriaareusingglucoseasacarbonsource.Astheglucosesourceisexhausted,thereisalaginthegrowthoftheculturewhiletheexpressionofgenesencodingenzymesnecessaryforlactosemetabolismareinduced.Then,inthelatterphaseofgrowth,lactoseisbeingmetabolized.

Time

Celldensity

SeealsoFigure7.3fornegativeregulation.

NegativeRegulation

promoter

lacZ

lacA

lacY

operator

repressor

monomer

repressor

tetramer

X

Notranscription

-lactose

lacI

POL

Transcriptionproceeds

+lactose

Inducer

allolactose

promoter

lacZ

lacA

lacY

operator

CAP

transcription

proceeds

lowglucose

ATP

cAMP

catabolite

activator

protein,CAP

+

POL

PositiveRegulation

promoter

lacZ

lacA

lacY

operator

notranscription

highglucose

cAMP

catabolite

activator

protein,CAP

X

X

CAP

lacZ

lacA

lacY

operator

ATP

promoter

-galactosidaseisanenzymethathydrolyzesthe-galactosidicbondinthedissacharide,lactose,toyieldthesimplesugars,glucoseandgalactose.GalactosidepermeaseisanenzymeneededtotransportlactoseintotheE.colicell.

BothnegativeandpositivecontrolofthelacoperonenablesE.colitouseavailablecarbonsourceswiththemaximumefficiency.Inordertoactivatethelacoperon,twoenvironmentalconditionsmustbemet.Firstly,lactosemustbepresentandthenegativecontrolsystemofthelacoperonrequireslactoseforde-repression.Thisavoidsthecostofsynthesizingenzymestometabolizeacarbonsourcethatisnotpresent.Thesecondenvironmentalconditionthatmustbemetforexpressionofthegenesofthelacoperon,isthatglucosemustnotbepresent.Lowglucoselevelsactivateapositiveregulatorypathwaythatinducestheexpressionofthegenesrequiredforlactosemetabolism.Thisensuresthatifbothglucoseandlactoseareavailable,thebacteriawillutilizethemostefficientcarbonsource,i.e.glucose.

SeeFigures7.6and7.7.NitrocellulosefilterbindingassaysusinglabeledlacoperatorDNAandpurifiedrepressorprotein,canbeusedtodemonstratethatthelacoperatoristhesiteofbindingfortherepressorprotein.Intheseexperiments,labeledoperatorDNAisboundtoincreasingamountsofrepressorproteinandthemixtureispassedthroughanitrocellulosefilter.OnlyDNAboundtoproteinisretainedonthefilterandwewouldexpectthedatatoshowincreasedamountoflabeledDNAastherepressorconcentrationincreases.ThegraphbelowillustratestheexpectedincreaseinDNA–repressorcomplexesinthepresenceofincreasedrepressoranditalsoshowsthatastheDNAbecomeslimitingthecurveplateaus.Asacontrolinthisexperiment,wecantesttheabilityofthelacoperontobindrepressorinthepresenceofanartificialinducer(IPTG).Wewouldpredictthatinthepresenceoftheinducertherepressorwillbeunabletobindtotheoperator.Thepredictedresultsareshownonthefigurebelow.

LabeledDNA

retainedonthefilter

Repressorconcentration

-IPTG

+IPTG

Toverifythatitistheoperatortowhichtherepressorisbindingintheseexperiments,wecanuseamutant(Oc)operator,whoseconstitutiveactivitysuggeststhatithasareducedaffinityforrepressor.AsFigure7.7demonstrates,ittakesaconsiderablyhigherconcentrationofrepressortoreachmaximalbindingwiththemutantoperator.Thus,whatisdefinedgeneticallyastheoperatorisreallythebindingsiteforrepressor.

SeeFigure7.8.ThefollowingexperimentcanbeusedtoshowthatRNApolymerasecanbindtothelacpromotereveninthepresenceoftherepressor.WeincubateDNAcontainingthelacpromoterwithrepressorproteinandallowtherepressortobindtotheoperator.Wethenaddbothinducerandrifampicin.Rifampicininhibitstheprocessofopenpromoterformation,butdoesnotinhibittranscriptionfromalreadyformedopenpromotercomplexes.Wewouldpredictthatiftheformationofanopenpromotercomplexrequiresreleaseoftherepressorfromtheoperator,transcriptionwouldnotoccurwhenrifampicinwasaddedsincetheopenpromotercomplexwouldnotbeforminginthepresenceoftheantibiotic.If,however,theRNApolymerasewasalreadyboundinanopenpromotercomplexpriortothereleaseoftherepressor,thenrifampicinwillnotaffecttranscriptionwhenaddedwiththerepressorasdescribed.Insuchanexperimentwewouldexpecttoobservetranscriptionafteradditionofinducerandrifampicin,supportingthehypothesisthatRNApolymerasecanbindtothelacpromoterinthepresenceoftherepressor.ThisistheresultthatFigure7.8depicts.

SeeFigure7.9.InvitrokineticstudiescanbeusedtoshowthatthelacrepressorpreventsRNApolymerasefrombindingtothelacoperon.Insuchanexperiment,alacpromoterisincubatedwithRNApolymeraseinthepresenceofaUTPanalogwithafluorescentlytagged-phosphate.Thereleaseoffluorescentlytaggedpyrophosphatecanbeusedtocalculatetherateofabortivetranscription.Abortivetranscriptionisaresultofrepeatedbindingofapolymerasetoatemplateandgenerationofshortoligonucleotidefragments.Inanexperimentsuchasthis,ifweaddheparin,therateofformationofabortivetranscriptswilldecreasebecausetheheparinwillbindthepolymeraseasitisreleasedfromthetemplate.Wecanhypothesizethat,iftherepressorisinterferingwithpolymeraserebindingtothelacoperon,thenadditionofrepressortotheassaywillalsoresultinareductionintherateofabortivetranscriptformation.AsanegativecontrolwewouldperformtheassayintheabsenceofDNAtemplate.Thegraphbelowshowstheexpectedresults.Theslopeofthelinesgivesustherateofabortivetranscriptformationaftertheadditionofheparinandafteradditionofrepressor.Asexpected,heparincompeteswiththepromoterforbindingofthepolymeraseleadingtoadecreaseinabortivetranscriptformation.Similarly,theobserveddecreaseintherateofabortivetranscriptformationaftertheadditionofrepressorisconsistentwiththerepressor’sinterferingwithbindingofthepolymerasetothepromoter.

SeeFigure7.11.Studiesofmutantlacconstructsinwhichoneormoreoperatorshavebeenremovedhavebeenusedtodemonstratethatallthreelacoperatorsarerequiredforfullrepression.M?ller-Hilletal.usedphagetointroducemutantconstructintoE.colistrainslackingalacZgene,andassayedthedegreeofrepressioninthelysogenicE.coli.Foldrepressioninthepresenceofeachintroducedgeneconstructwasmeasuredbycomparingthe–galactosidaseactivityinthepresenceandabsenceoftheinducerIPTG.Mutatingoneortheotheroftheauxiliaryoperatorsshowedlittleeffectonrepression.However,removalofbothauxiliaryoperatorsresultedina50-foldlossofrepression.

Aconvenientassaythatallowsustomeasurethestimulationof–galactosidaseactivitybyadditionofcAMP,wasdevelopedbyZubayandco-workers.AcellfreeextractofE.coliispreparedandthe–galactosidaseactivityintheextractcanbemeasuredaftertheadditionofdifferentamountsofcAMP.Astimulationofthe–galactosidaseactivityuponcAMPadditionisconsistentwiththepresenceofaprotein,CAP(cataboliteactivatorprotein),intheextractthatuponbindingtocAMPactivatesthelacoperon.E.colimutantsexistthathaveaCAPproteinwithareducedaffinityforcAMP.WecanusecellfreeextractsfromthesemutantE.coliandmeasurethe–galactosidaseactivityintheseextractsafteradditionofincreasingamountsofcAMP.Wewouldpredictthat,becauseofthemutantCAPpresentinthecellfreeextracts,wewouldobserveadecreasecAMPstimulated–galactosidaseactivity.Expecteddataareshowninthefigurebelow.

TheCAP-cAMPcomplexfunctionsinanumberofwaystoactivatetranscriptionfromthelacpromoter.First,CAP-cAMPindirectlystimulatestheformationofopenpromotercomplexes.Thisisbecauseitstimulatestheformationofclosedpromotercomplexeswhichincreasestheopportunityforopenpromotercomplexformation.CAP-cAMPalsoreducesinefficientinitiationoftranscriptionthatoccursatanalternativepromotersite,P2,withinthelacoperon.ThishastheeffectofincreasingtheavailabilityofRNApolymerasemoleculesforformingopenpromotercomplexesatP1,whichistheprimaryefficientpolymerase-bindingsite.Onceboundtotheactivator-bindingsiteupstreamofthepolymerase-bindingsite,CAP-cAMPinteractsdirectlywithRNApolymerase.Specifically,ActivatorRegionI(ARI)ofCAP-cAMPinteractswiththeCarboxy-terminaldomainofthe-subunitofRNApolymeraseI(-CTD)(Figure7.19).TheinteractionbetweenARIand-CTDleadstoco-operativebindingofRNApolymerasetothepromoter.Finally,CAP-cAMPbendsitstargetDNAuponbindingandthisstimulatestranscriptionbyfacilitatinginteractionswiththeRNApolymerase.

SeeFigure7.18.ThefollowingelectrophoresisexperimentcanbeusedtodemonstratethatbindingoftheCAP-cAMPcomplextothelacpromoterregionbendstheDNAmolecule.InsuchanexperimentwewouldtakeadvantageofthefactthataDNAfragmentthatisbentwillhavealowerelectrophoreticmobility.Thelocationofthebendaffectsthedegreeofretardationonagel.Inotherwords,twoproteinmoleculesofthesamesizeboundtothesamepieceofDNA,willdifferintheirmobilitybaseduponwherewithintheDNAmoleculetheproteinisbound.Aproteinboundinthecenterwillaffectthemobilitymorethanoneboundofftotheside.WecanuserestrictionenzymedigestiontogenerateDNAfragmentsofthesamesize,allofwhichcontaintheCAP-cAMPbindingsiteatadifferentbutknownlocation.WewouldthenbindtheCAP-cAMPcomplextotheDNAandwecanpredictwhichDNAfragmentwillhavethelowestmobility.WeexpectthatwhenthebindingsiteislocatedinthecenterofthefragmentthemobilitywillbelowestandinassaysusingtheconstructswiththebindingsiteoffcenterwewillseeadecreaseintheobservedretardationofmobilityasafunctionofitsdistancefromthecenteroftheDNAfragment.WewouldexpecttheDNAfragmentwiththelowestmobilitytohavetheCAP-cAMPbindingsiteatthecenteroftheDNAfragment,thussupportingthehypothesisthatbindingoftheCAP-cAMPcomplextothelacpromoterregionbendstheDNAmolecule.Figure7.18showsthatthisexpectationwasborneoutbyexperiment.

SeeFigure7.17.X-raycrystallographyhasbeenusedtoconfirmthepresenceofabendintheDNAmoleculeatthelacpromoterregioninresponsetobindingoftheCAP-cAMPcomplex.

SeeFigure7.20.RepressionofthearabinoseoperonrequiresbindingofAraCtoboththearaO2andthearaIsitesinthearaBADoperon.Thesetwobindingsitesareseparatedbyover200hundredbasepairsandloopingoutoftheinterveningDNAsequencesisrequiredtoallowtheseproteinstointeract.TheirinteractioniscontingentupontheirbeingalignedwitheachotheronthecorrectfaceoftheDNA.Inotherwords,aftertheloopisformedeachmustbeonasideofthehelixthatallowsthemtointeract.InsertionofaDNAfragmentthatintroducesoneormoreentirehelicalturnswillnotinterferewithprotein-proteininteractionsbecauseitwillnotaltertheirpositionsontheDNAfacerelativetoeachother.Ontheotherhand,ifanon-integralnumberofturnsareintroducedbetweenthetwoproteinbindingsitestheproteinswillfindthemselvesondifferentfacesoftheDNAafterformationoftheloop.Interactionbetweentheproteinscannotoccursincethehelixcannottwistaroundtoallowtheproteinstocontacteachother.Thediagrambelowillustratesthisphenomenon.OntheleftofthediagramanintegralnumberofDNAhelicalturnshavebeenintroducedbetweenthetwoproteinbindingsitesandontherightanon-integralnumberofturnshasbeenintroducedandconsequentlytheproteinsareunabletointeract.

SeeFigure7.21:

AraC+arabinose

AraC

SeeFigure7.23.InordertodemonstratethatarabinosecanbreaktherepressionloopformedbyAraC,wecanuseelectrophoresistodetecttheformationoftheloopinthepresenceandabsenceofarabinose.Todothisexperiment,wewouldprepare“minicircles”ofDNAofaround400basepairscontainingthearaO2andthearaIsitesseparatedby160basepairs.TodetectloopformationinthisconstructwecantakeadvantageofthefactthattheformationofaloopinasupercoiledpieceofDNAwillincreaseitselectrophoreticmobility.Wewouldthenlabeltheseminicirclesandincubatetheminthepresenceandabsenceofarabinose.WewouldthenelectrophoresetheDNAandweexpecttoseeanincreaseinthemobilityoftheDNAintheabsenceofarabinoseduetothepresenceoftherepressionloop.However,inthesamplewitharabinosewewillseelowermobilityoftheDNAbecausethearabinosehasinhibitedrepressionloopformation

SeeFigure7.22,TodemonstratethatbotharaO2andaraIareinvolvedinformingtherepressionloopwecanuseminicircleassaysusingmutantconstructs.Theprinciplebehindtheseexperimentsisasfollows.IfthestabilityoftherepressionlooprequiresbindingofAraCtobotharaO2andaraI,disruptingthebindingofAraCtoeitherofthesesitesbymeansofamutationintheDNAsequencewillreducethestabilityoftherepressionloop.Wewouldsetuptheexperimentinthefollowingmanner.WewouldbindAraCtolabeledminicirclescontainingeitherwild-typebindingsites,orbindingsitescarryingamutationateitheraraO2oratbotharaIbindingsites.Thenwewouldaddunlabeledwild-typeminicircles,performelectrophoresis,andmeasurethetimeittakesforhalfoftherepressionloopstodissociate.Wecanmeasurelossofthelabeledrepressionloopsbylookingfordecreaseinmobilityonagel.Themorerapidlythecomplexesdissociate,thelessstabletherepressionloop.WeexpecttoobservethatminicircleswithmutationsineitheraraO2orintheara1bindingsitesaremorereadilyconvertedtounloopedcirclesthanarethewild-typeminicircles.FromthiswecaninferthebothofaraO2andariIarerequiredforrepressionloopformation.

SeeFigure7.24.Todemonstratethatremovingarabinosefromrepressionloopsallowsthemtoreform,wewouldperformthefollowingexperiment.Wewouldgeneraterepressionloopsasdescribedaboveandwewouldaddarabinosetoasubsetofthereactionspriortoelectrophoresis.WewillobserveloweredmobilityoftheDNAcomplexreflectingbreakdownoftherepressionloops.Ifwethentaketheremainingreactionmixtureanddiluteitwithbuffer,wewillbeeffectivelydecreasingtheconcentrationofarabinoseandwewillobservereformationoftherepressionloopsuponelectrophoresis.

TwoexperimentsareneededtodemonstratethataraI2isimportantinbindingAraCwhentheDNAisintheunloopedformbutnotinloopedform.Thefirstexperimentwesetupwouldusemethylationinterference.TheprinciplebehindthisapproachisthatregionsoftheDNAthatbindAraCandthatareimportantforloopingwillbeinhibitedfromloopingwhentheyaremethylated.Therefore,inapartiallymethylatedsetofDNAmoleculesthatwereloopedaftermethylation,wewouldexpectonlytheDNAthatisunmethylatedincriticalregionstobelooped.ToperformthisexperimentwewouldpartiallymethylateminicircleDNAsuchthatonaverageonlyonebaseorasmallnumberofbasespermoleculeweremethylated.WewouldthenaddAraCtolooptheDNAandseparatetheloopedDNAfromtheunloopedDNA.IfaregionofDNAwereresponsibleforloopingwhenitboundAraCwehaveeffectivelyselectedagainstmoleculesmethylatedatthisregionandwewouldexpecttoonlyfindloopedmoleculesthatwerenotmethylatedatthissite.Ifweanalyzethemethylationpatternsoftheloopedmoleculesafterthispartoftheexperiment,weexpecttofindaraI1basesthatareheavilymethylatedintheunloopedDNAbutunmethylatedintheloopedDNA.However,whenwelookatthemethylationinthearaI2regionofloopedandunloopedDNAwewillfindmethylatedDNAinbothsamplesindicatingtheAraCdoesnotcontactaraI2intheloopedstate.Wewouldsetupasecondexperiment(Figure7.25)todemonstratethatara12isimportantinbindingAraCwhentheDNAisintheunloopedstate.Thetoolsforthisexperimentaregelshiftassaysusingbothwild-typeandmutantaraI2minicircles.InthisexperimentwewouldfirstbindAraCtobothwild-typeandmutantaraI2minicircles.WewouldthenbreaktheloopandlinearizethecircleswitharestrictionenzymeanddetermineifAraCisboundtothemutantandthewild-typearaI2.WeexpecttoseealossofbindingintheminicirclewiththemutantaraI2site.ThistellsusthataraI2isnecessaryforbindingAraCintheunloopedstate.

SeeFigure7.27.ThegenesofthetrpoperoninE.colicellsareinactivatedinresponsetoabundanttryptophan.Amodelforthisnegativecontrolmechanismisasfollows.Anaporepressormonomerexistsinthecellandithasnoaffinityforthetrpoperonunlessitisassociatedwithacorepressor.Thecorepressoristryptophan,anditsassociationwiththeaporepressorresultsinanallostericchangeallowingtheformationofanactiverepressorcomplex.Thiscomplexbindsthetrpoperatorasadimer,repressingexpressionofthetrpstructuralgenes.Thus,whentryptophanisabundantinthecell,thegenesofthetrpoperonarerepressedandwhenitisscarce,theoperonisreleasedfromrepression.

SeeFigures7.28and7.29.Attenuationinthetrpoperonisfacilitatedbyanattenuatorwithinaleadersequenceupstreamofthefirststructuralgeneinthetrpoperon.TheattenuatoriscomposedofaninvertedrepeatintheDNAsequence,followedbyastringofT’sinthenon-templatestrand.Inthepresenceoftryptophan,theRNAmoleculeformsahairpinloopbecauseoftheself-complementarityoftheinvertedrepeats.ThetractofU’s,whichmakeunstableUAbasepairswiththetemplate,combinewiththehairpinlooptomakethetranscript/templatehybridunstable.Thisresultsinterminationoftranscriptionandreleaseofthetranscript.

Translationofthetrpleaderregiondoesnotcontinueintothetrpstructuralgenesbecausethereisatranslationterminationsequenceattheendofthetrpleadersequence.

SeeFigure7.32.Whentryptophanisscarce,attenuationofthetrpoperoncanbeoverridden,allowingexpressionofthestructuralgenesintheoperon.InspectionoftheDNAsequencesintheleadersequencerevealssomefeaturesthatarekeytothecapacityofRNApolymerasetobypasstheattenuationsequencewhentryptophanlevelswithinthecellarelow.WithintheleadersequencethereareinfacttwoinvertedrepeatsthatwillresultinRNAcontainingtworegionscapableofhairpinloopformation.ThesecondordownstreamhairpinloopisassociatedwithatractofU’s.Thisisthefunctionalattenuator.ThefirstorupstreamhairpinloopdoesnothavetheoligoUtractassociatedwithitanditdoesnotfunctionasanattenuator.Theleadersequencecanformacompositehairpinloopusingasinglerepeatfromeachofthetwohairpinloops.ThisalternativehairpinisnotassociatedwithapolyUtractandthereforewillnotleadtoattenuation.Formationofthisalternativehairpinloopprohibitstheformationofthedownstreamhairpinloopthatisneededforattenuation.Alternativehairpinloopformationisfavoredwhentheribosomestallsattwotryptophancodonsupstreamoftheinvertedrepeats.Thisstallingoccursundersituationswheretryptophanconcentrationsarelow.Thus,whentryptophanislimiting,transcriptioncanproceedthroughtothestructuralgenes.

SeeFigure7.33.AriboswitchisaregionofRNAsequencewithinthe5’UTRofanmRNAthatcontrolsthesynthesisortranslationofthatRNA.Thisisachievedbythebindingofasmallmolecule,orligand,toaspecificregionoftheriboswitchcalledanaptamer.AnexampleistheribDoperonofBaccillussubtilus.Aconservedelement,theRFNelement,bindsFlavinMononucleotide(FMN)aproductoftheribDoperon.ItisbelievedthatbindingofFMNtotheaptamercausesaconformationalchangeintheelement,resultingintheformationofaterminator.Inthiswaytheproductoftheoperonswitchesoffitsownproduction.

In-lineprobingisatechniquethatallowsustoprobethestructureofanRNAmolecule.SpontaneouscleavageofRNAmoleculesoccursmorereadilyintheabsenceofsecondarystructure.Thisisbecausehydrolysisneedsa180arrangementbetweentheattackingnucleophileanditssubstrate,andsuchanarrangementispreventedinbase-pairedregionsofanRNA.Thisallowsustovisualizechangesinthesecondarystructurethatmayoccuruponbindingofaligandtoitsaptamer.SeparatingthecleavedfragmentsonagelandobservingafootprintontheautoradiographallowsvisualizationofanyregionoftheRNAthatislesssusceptibletocleavage.ThisisanalogoustoafootprintwewouldobserveinaDNasefootprintingexperiment.Todoanin-lineprobingexperiment,anRNAfragmentcontainingtheaptamerislabeledandthenincubatedinthepresenceandabsenceofitsligand.Spontaneouscleavagepatternareobservedbyelectrophoresisandautoradiography.AsetoffragmentsontheautoradiographthataremissingonlyinthepresenceofligandsuggeststhattheligandisboundtothissiteintheRNAinducingachangeinitssecondarystructure.

AnalyticalQuestions

phenotypeminusinducer

phenotypeplusinducer

Explanation

a

-

+

Allthegenesarewild-typethereforewild-typefunctionispresentand-galactosidaseisproduceduponinductiononly.

b

-

+

TheZ-mutantalleleisrecessivetotheZ+allele.Theenzymeproducedbythewild-typegeneissufficienttogiveawild-typephenotype.

c

-

+

TheI-mutantalleleisrecessivetotheI+allele.Therepressorproteinproducedbythewild-typeallelewillbesufficienttobindtheoperatorinbothgenes.Inotherwords,themutationwillbecomplementedintransbythesinglewild-typeallele.

d

-

-

TheIsmutantalleleisdominantovertotheI+allele.RepressorproteinsproducedfromtheIsareunabletobindtheinducerandthuscannotbereleasedfromtheoperator.Therepressorproducedinthemerodiploidwillcontainsubunitsfromthemutantalleleaswellasthewild-type.Thiswillrenderthetetramernon-functionalanditwillbepermanentlyassociatedwiththeoperator.

e

+

+

TheOcmutantalleleisdominantovertotheO+allele.Themutantoperatorisunabletobindrepressorproteinandwillthereforeconstitutivelyproduce-galactosidaseproteininitsoperonregardlessofthepresenceofabsenceofinducer.

f

-

+

SincetheOcmutationisinthepromotercontrollingexpressionofamutantlacZgene,themutationinthelacZgenegeneis“cancelingout”theOcmutation.TheZ-alleleisepistaticontheOcallele.

g

+

+

SincetheOcmutationrendersthepromoterincapableofbindingtherepressor,it“cancelsout”thephenotypeoftheIsallelewhichproducesrepressorthatisunabletobindtheinducer.TheOcalleleisepistaticontheIsalleleandthisoperatorwilldriveconstitutiveexpressionofthe-galactosidasegene.Thewild-typeoperatorwillalsonotbedepressiblebecausetheIsalleleisdominantovertheI+alleleforthereasonsexplainedind.above.However,sincetheothercopyofthelacZgeneisproducing-galactosidaseconstitutivelytherewillbe-galactosidaseproducedinthepresenceoftheinducer.

(a)Becausethephenotypeofmutantstrain#1isconstitutive(noinducerneededforactivity),itsgenotypemustcarryamutationineithertheIortheOgene,i.eI-orOc.Therefore,C(themutantgeneinstrain#1)iseitherIorO.Mutant#2isalsoconstitutive,soitsgenotypeisalsoeitherI-orOc.Therefore,AiseitherIorO.SofarweknowthatAandCareIandO,butnotnecessarilyinthatorder.ThismeansthatBmustcorrespondtoZ,byelimination.NowweneedtodeterminewhetherA=IandC=O,orvice-versa.ThephenotypeofMutant#3givestheanswer.Becauseitisconstitutiveandthemutantformisdominant,C-mustbeOc.IfitwereI-,theotherconstitutivepossibility,themutationwouldhavebeenrecessive,andno-galactosidasewouldhavebeenproducedintheabsenceofinducer.ThusA=I,B=Z,andC=O.

(b)Thepartialdiploidinline4failstomake-galactosidaseeveninthepresenceofinducer.Thatis,themutation(A-)isdominant,andA-mustthereforebeIs.Ontheotherhand,thephenotypeofthestraininline5isthesameasthatofawild-typestrain.Therefore,themutation(A-)inthegenotypeofthestrainshowninline5mustberecessive,i.e.A-=I-.Therefore,thetwogenotypesare:

IsO+Z+/I+O+Z+

I-O+Z+/I+O+Z+

E.colicellswithamutantlacoperatorthatisunabletobindtherepressor,willexpressthegenesfromthelacoperonconstitutively.Inotherwords,thegeneswillbeexpressedinthepresenceandabsenceoflactose.

E.colicellswithamutantlacrepressorthatcannotbindtothelacoperatorwillexpressthegenesfromthelacoperonconstitutively.Inotherwords,thegeneswillbeexpressedinthepresenceandabsenceoflactose.

E.colicellswithamutantlacrepressorthatcannotbindtoallolactosewillbeu