Lehninger-Principles-of-Biochemistry-习题答案chapter-2

RegulationofGeneExpressionS-3101.EffectofmRNAandProteinStabilityonRegulationE.colicellsaregrowinginamediumwithglucoseasthesolecarbonsource.Tryptophanissuddenlyadded.Thecellscontinuetogrow,anddivideevery30min.Describe(qualitatively)howtheamountoftryptophansynthaseactivityinthecellschangesunderthefollowingconditions:(a)ThetrpmRNAisstable(degradedslowlyovermanyhours).(b)ThetrpmRNAisdegradedrapidly,buttryptophansynthaseisstable.(c)ThetrpmRNAandtryptophansynthasearebothdegradedrapidly.AnswerThemRNAfromthetrpEDCBAoperonencodesseveralenzymesfortryptophanbiosynthesis;thetrpBandtrpAgenesencodetryptophansynthase.ThecompletetrpmRNAissynthesizedonlywhentheconcentrationoftryptophan(actually,thatofchargedTrp-tRNA)islow.Thisistheresultofrepressionandattenuation(seeProblem6).Thereisnostrongregulationattranslation,sowhentrpmRNAispresent,tryptophansynthaseisproduced.Althoughtheregulationoftryptophanbiosynthesis,likemostbiosyntheticpathways,isfine-tunedbyfeedbackcontrol,feedbackinhibitionbytryptophanisexertedatthebranchpointanthranilatesynthase(theproductofthetrpEandtrpDgenes;seeFig.28–19),sotheactivityoftryptophansynthaseisnotstronglyaffectedby[tryptophan].(a)IfthetrpmRNAisstablerelativetocellgenerationtime,itpersistsinthepopulationofbacteriaevenaftertryptophanhasbeenadded,andtryptophansynthasecontinuestobesynthesizedandactive.Inasimplemodel,wewouldexpecttoseetheenzymeactivitypercellroughlyhalvedforeachgeneration(30min);thatis,theactivitywouldbeslowlydilutedoutbytheincreasingnumbersofcells.(b)Again,iftheenzymeisstablerelativetothegenerationtime,itpersistsinthepopulation,evenaftertheadditionoftryptophan,andremainsactive.(c)IfthemRNAandenzymeareunstable(degradedrapidlyrelativetocellgenerationtime),attenuationoftranscriptionofthetrpoperoncausedbyadditionoftryptophanleadstoanabruptdecreaseinlevelsoftrpmRNAandtryptophansynthase.2.NegativeRegulationDescribetheprobableeffectsongeneexpressioninthelacoperonofamutationin(a)thelacoperatorthatdeletesmostofO1;(b)thelacIgenethatinactivatestherepressor;and(c)thepromoterthatalterstheregionaroundposition10.AnswerThelacoperonisnegativelyregulatedbyarepressor,theproductofthelacIgene.TheLacrepressorbindstospecificDNAsequencescalledtheoperators(O1andpseudo-operatorsO2andO3).BindingoftherepressorpreventsefficientinitiationoftranscriptionbyRNApolymerasefromthepromoter.Aninducer(allolactoseorananalog)bindstotherepres-sorandpreventsitsbindingtotheoperator,therebyrelievingtherepressionandallowingtranscriptionofthelacoperon.chapter282608T_ch28sm_S310-S31802/26/200812:20amPageS-310pinnacleOS9:DesktopFolder:WHQY028:(a)Thismutationintheoperator,thebindingsiteforrepressor,wouldlowertheaffinityfortherepressorandhencereducerepressorbinding.Thiswouldallowcontinuedtranscrip-tion(thusexpression)ofthelacoperonevenintheabsenceofinducer—referredtoasconstitutiveexpression.(b)AmutationinthelacIgenethatproducedarepressorunabletobindtotheoperatorwouldleadtoconstitutiveexpression(norepressionintheabsenceofinducer).Amuta-tionthatpreventedbindingoftherepressortotheinducerwithoutaffectingtheabilitytobindtotheoperatorwouldleadtoanoninduciblephenotype(constantrepression).(c)Theregionofthelacgenearoundposition10isthepromoterregion.Thelacpro-moterisnotparticularlystrong.Mutationshavebeenobservedthateitherincreaseordecreaseitsefficiencyofinitiatingtranscription.Basesubstitutionsthatmakethepro-motersequencemoresimilartotheconsensusgenerateastrongerpromoter(promoter“up”mutations);thosethatmakethepromoterlesssimilartotheconsensusgenerateaweakerpromoter(promoter“down”mutations).An“up”mutationwouldmakethelacoperonindependentofpositiveregulationbythecAMP-CAPcomplex(whentheoperonisinduced).A“down”mutationwouldnotallowexpressioneveninthederepressedstate(presenceofinducer)andhencewouldproduceanoninduciblephenotype.3.SpecificDNABindingbyRegulatoryProteinsAtypicalbacterialrepressorproteindiscriminatesbetweenitsspecificDNAbindingsite(operator)andnonspecificDNAbyafactorof104to106.About10moleculesofrepressorpercellaresufficienttoensureahighlevelofrepression.Assumethataverysimilarrepressorexistedinahumancell,withasimilarspecificityforitsbindingsite.Howmanycopiesoftherepressorwouldberequiredtoelicitalevelofrepressionsimilartothatinthebacterialcell?(Hint:TheE.coligenomecontainsabout4.6millionbp;thehumanhaploidgenomehasabout3.2billionbp.)AnswerThediscreteDNA-bindingdomainsoftranscriptionalregulatoryproteinsformspe-cificcomplexeswithdefinedsequencesofDNA.Theiraffinityforthesedefinedsequencesisabout104to106greaterthantheiraffinityforothersequences.UsingtheexampleoftheLacrepressor,thebindingsite(operator)is22bplong.TenmoleculesoftheLacrepressoraresufficienttokeepthisoperatorinaboundstate,eveninthecontextof4.6106bpofnonspecificDNA(therestoftheE.coligenome).Thisamountstofindingonespecificsiteinaseaof(4.6106bp)/(22bp)2.1105nonspecificsites.Forthehypotheticalrepressorinahumancell,letususethesamesizebindingsite(22bp),althoughthisislargerthanmostsitessofarcharacterized.Thehumanrepressormustfinditsspecificsitewithin(3.2109bp)/(22bp)1.5108nonspe