chapter12GeneRegulation.

分子生物学系列讲座之6:ZhangjieMobile:13673550700Email:[e-mail]17:28:182主要内容GenelevelTranscriptionlevelPost-transcriptionlevelTranslationlevelpost-translationlevel17:28:18312.1TermsPositivecontrol(正调控)Negativecontrol(负调控)Regulatoryproteincis-actingelement(顺式作用原件)trans-actingfactor(反式作用因子)operator(操纵子)图11-1正调控与负调控模式的比较17:28:184TermsStructuralgene(结构基因)Regulatorgene(调节基因)constitutiveprotein(组成型蛋白)regulatorprotein(调节蛋白)Activator(激活蛋白)Aporepressor(阻遏物蛋白)corepressor(辅阻遏物)17:28:185Inducible(可诱导)repressible(可抑制)repressor(阻遏物)activatorprotein(AP)(激活蛋白)inducer(诱导物)corepressor(辅阻遏物)silence17:28:186FIGURE12.27Controlcircuitsareversatileandcanbedesignedtoallowpositiveornegativecontrolofinductionorrepression.17:28:18712.1.1DNA水平上的调控组成型启动子:与一致序列相同或相近，恒定的转录速率，不受其他形式的调控弱启动子:缺乏一个或多个一致序列元件，低转录速率，经常需激活蛋白强启动子:与一致序列相近或相同，高转录速率，经常受到阻遏17:28:188图11-2鼠伤寒沙门氏菌相变的分子机制GeniclevelcontrolDNA重组对基因表达调控17:28:18912.2操纵子学说Theoryofoperon1961-196512.2.1Firstexample:LacoperonRegulationofTranscriptionInitiationinBacteriaThelactoseOperon(乳糖操纵子)17:28:1811Promoter(启动子)Operator(操纵子)Structuralgene(结构基因)regulatorygene(调节基因)Inducibleoperons(可诱导操纵子)Repressibleoperons(可抑制操纵子)17:28:1812图11-5β-半乳糖苷酶催化的水解和异构化反应操纵子的发现1961年Jacob&Monod构建部分二倍体(lacI-/FlacI+)lacIS阻遏蛋白不可诱导性突变，阻遏蛋白失去诱导物结合位点lacI-阻遏蛋白不能形成寡聚物lacI-d阻遏蛋白不能和DNA结合，且呈负互补(反式显性)Oc操纵基因的组成型突变FIGURE12.6AdditionofinducerresultsinrapidinductionoflacmRNA,andisfollowedafterashortlagbysynthesisoftheenzymes;removalofinducerisfollowedbyrapidcessationofsynthesis.表16-1在单倍体和部分二倍体中β-半乳糖苷酶透过酶的合成基因型β-半乳糖苷酶透过酶不诱导诱导不诱导诱导I+Z+Y+－＋－＋I–Z+Y+＋＋＋＋I+Z–Y+/FI–Z+Y+－＋－＋I–Z–Y+/FI+Z+Y+－＋－＋I–Z–Y+/FI–Z+Y-＋＋＋＋△(I,Z,Y)/FI–Z+Y+＋＋＋＋O+Z+Y+－＋－＋O+Z+Y+/FO+Z–Y+－＋－＋OcZ+Y+＋＋＋＋O+Z+Y-/FOcZ+Y+＋＋＋＋O+Z+Y+/FOcZ–Y+－＋＋＋O+Z–Y+/FOcZ+Y-＋＋－＋IsO+Z+Y+/FOcZ+Y+＋＋＋＋△:表示缺失，“＋”表示酶以最高水平存在，“－”表示酶以最低水平存在Lactoseoperon(lac)FIGURE12.4Thelacoperonoccupies-6000bpofDNA.AttheleftthelocIgenehasitsownpromoterandterminator.TheendofthelocIregionisadjacenttothepromoter,P.Theoperator,0,occupiesthefirst26bpofthetranscriptionunit.Thelonglaclgenestartsatbase39,andisfollowedbythelacYandlacAgenesandaterminatolacYencodesacellmembraneproteincalledlactosepermease(半乳糖苷渗透酶)totransportLactoseacrossthecellwalllacZcodesforβ-galactosidase(半乳糖苷酶)forlactosehydrolysislacAencodesathiogalactosidetransacetylase(硫代半乳糖苷转乙酰酶)togetridofthetoxicthiogalacosides17:28:1819图11-6lac操纵子的负调控17:28:1820FIGURE12.7Repressormaintainsthelacoperonintheinactiveconditionbybindingtotheoperator.Theshapeoftherepressorisrepresentedasaseriesofconnecteddomainsasrevealedbyitscrystalstructure(seelater).17:28:1821FIGURE12.8Additionofinducerconvertsrepressortoaninactiveformthatcannotbindtheoperator.ThisallowsRNApolymerasetoinitiatetranscription.17:28:1822FIGURE12.5RepressorandRNApolymerasebindatsitesthatoverlaparoundthetranscriptionstartpointofthelacoperon.17:28:1823CH2OHCH3HOOS－C－CH3HCH3OHHHHHOH图16-6异丙基-β-硫代半乳糖苷的分子结构17:28:1824图11-7乳糖操纵子三个阻遏蛋白结合位点的结构特征及其作用激活因子:CAP(CataboliteActivatorProtein,代谢产物激活蛋白)orCRP(cAMPReceptorProtein,cAMP受体蛋白);responsestotheglucoselevel.cAMP(腺苷-3',5'-环化一磷酸)腺苷酸环化酶促进cAMP↑磷酸化的ⅡAGlu使腺苷酸环化酶↑葡萄糖通过PTS(磷酸转移系统)进入细胞使磷酸化的ⅡAGlu↓17:28:1826NH2P~P~P－O－CH2ATPOHOHGlucoseDNAAdenylcyclaseIhibition?RNApol+cyclicAMP+proteinfactorneededforNH2transcriptiontotackplaceO－CH2“X”ProteinfactorCyclicAMP(catabolite)RNAPOOHStimulationPhosphodiesteraseNH2P－O－CH25’AMPOHOH图16-Controlofcatabolite-sensitivetranscriptionthroughcyclicAMPFIGURE12.29ByreducingthelevelofcyclicAMP,glucoseinhibitsthetranscriptionofoperonsthatrequireCRPactivity.17:28:1828图11-8乳糖操纵子的操纵基因和CAP-cAMP结合位点序列17:28:1829图11-9CAP的正调控作用17:28:18305.CAPandLacrepressorbindDNAusingacommonstructuralmotif:helix-turn-helixmotifOneistherecognitionhelixthatcanfitsintothemajorgrooveoftheDNA.17:28:1831图11-10CAP-cAMP与其结合位点结合以后导致DNA发生的弯曲17:28:1832图11-11CAP-cAMP在CAP位点上与RNApol的相互作用17:28:1833FIGURE12.32TheeRPproteincanbindatdifferentsitesrelativetoRNApolymerase.CAP结合位点位于启动子内,gal与启动子相邻,lac位于启动子上游,ara色氨酸操纵子结构:trpA\B\C\D\E特点:(1)trpR(89’)和trpABCDE(25’)不连锁；(2)操纵基因在启动子内(3)有衰减子(attenuator)(4)启动子和结构基因不直接相连，二者被前导顺序(Leader)所隔开分支酸→邻氨基苯甲酸→磷酸核糖基→CDRP→吲哚甘油-磷酸→色氨酸邻氨基苯甲酸邻氨基苯甲酸合成酶吲哚甘油色氨酸合成酶硼酸合成酶β链α链60，00060，00045，00050，00029，000POlatrpEtrpDPtrpCtrpBtrpAtt’156016201353119180436P：起动子；O：操纵子；l：前导序列；a：衰减子；t,t’：终止子图16-15E.colitrpO的结构及其产物所催化的色氨酸合成反应trp操纵子的结构trpR17:28:1838图11-18色氨酸操纵子的阻遏调控trpRtrpPtrpOtrpEtrpDtrpCtrpBtrpA蛋白TrpR(无活性)活化的阻遏蛋白阻遏物(Trp)图16-27TrpR被Trp激活后可阻遏trp操纵子的转录(仿B.Lewin:《GENES》Ⅳ，1990,Fig.13.16)过量3.调节(1)Trp作为辅阻遏物(corepressor)(2)阻遏物和RNApol在P,O重叠区产生竞争性抑制；(3)阻遏物的阻遏能力低，是LacR的1/千；(4)trpO调节合成代谢，存在衰减作用。转录的终止调控衰减作用1978年yanofsky发现前导序列(leadersequence)衰减子（attenuator）17:28:1842TheleaderRNAandleaderpeptideofthetrpoperonFIGURE13.6Thetrpoperonconsistsoffivecontiguousstructuralgenesprecededbyacontrolregionthatincludesapromoter,operator,leaderpeptidecodingregion,andattenuator.17:28:1844FIGURE13.8Thetrpleaderregioncanexistinalternativebase-pairedconformations.Thecentershowsthefourregionsthatcanbasepair.Region1iscomplementarytoregion2,whichiscomplementarytoregion3,whichiscomplementary