分生-课件分子生物学13-regulation of transcription in prokaryotes

RegulationoftranscriptioninprokaryotesTheOperons

TheLACOperon(乳糖操纵子)

TheTRPOperon(色氨酸操纵子)

AlternativeSigmafactors

（利用不同的sigma因子进行转录的调控）Whatistheoperon?Theoperonisaunitofbacterialgeneexpressionandregulation,includingstructuregenesandcontrolelementsinDNArecognizedbyregulatorygeneproduct(s).操纵子是一套原核基因表达和调控的单元，它包括被协同调节的基因和被调节基因的产物所识别的调控元件。Atypicaloperonincludes：

1.

Structuralgenes（结构基因）

2.Controlelement（调控元件）

3.Regulatorgene（调节基因）

promoter

structural

genes

operatortranscription,translationandproteinsynthesisbindingsiteforregulatorproteinsbindingsiteforRNApolymeraseKeyTermsNegativeControlPositiveControlUndernegativecontrol,geneticexpressionoccursunlessitisshutoffbysomeformofaregulatormoleculeUnderpositivecontrol,transcriptionoccursonlyifaregulatormoleculedirectlystimulatesRNAproductionThelacOperon（乳糖操纵子）Z：b-galactosidaseY：permeaseA：transacetylasePolycistronicmRNA:containingmorethanonecodingregionunderthesameregulatorycontrol.多顺反子信使RNA（即：多基因信使RNA）AUGUAAAUGUAGAUGUAACistron1Cistron2Cistron3AUGUAAAAAAAAAAAPolycistronandsinglecistron单顺反子singlecistronlacRepressorlac阻抑物-aproductofthelacI

gene基因LacI的产物tetramerof4identicalpolypeptides四聚体Bindstheoperator与操纵序列结合PreventsRNApolymerasefrombindingtothepromoter

阻止RNA聚合酶与启动子结合

Therefore,whentherepressorbindstheoperator,operonisrepressed.

当lac阻抑物与操纵序列结合时，操纵子是受阻抑的。Aslongasnolactoseisavailable,lacoperonisrepressed

一旦无乳糖存在，乳糖操纵子是受抑制的。lacrepressortetramersbindtoDNABlue-DNAred,yellow,green,andpink–4monomersofrepressorfrontsideβ-galactosidasefunctions

半乳糖苷酶的功能1.Cleavageoflactosetogalactoseandglucose

将乳糖分解成半乳糖和葡萄糖乳糖半乳糖葡萄糖Allolactose（异乳糖）isanaturalinducer

Lactose（β-1，4linkage）Allolactose（β-1，6linkage）β-galactosidasefunctions

半乳糖苷酶的功能2.催化乳糖形成异乳糖.

Allolactoseisanaturalinducer异丙基-β-D-硫代半乳糖苷5-溴-4-氯-3-吲哚-β-D-半乳糖苷Itisasubstrateofβ-galactosidaseItisnotasubstrateofβ-galactosidaseControlofthelacOperon

乳糖操纵子的调控Thelacoperonistightlycontrolled,using2typesofcontrolNegativecontrol,likethebrakeofacar,mustremovetherepressorfromtheoperatorPositivecontrol,anactivatorrespondstolowglucosebystimulatingtranscriptionofthelacoperon.

乳糖操纵子是在严格的调控之下的，有两种调控类型：--负调控，象刹车，必须从操纵基因上移去阻抑物。--正调控，一个激活因子，在葡萄糖浓度低时刺激乳糖操纵子的表达。Induction（诱导）

TherepressorisanallostericproteinBindingofonemoleculetotheproteinchangesshapeofaremotesiteonthatproteinAlteringitsinteractionwithasecondmoleculeInducer(onemolecule)oflacoperonbindstherepressor,causingtherepressortochangeconformationthatfavorsreleasefromtheoperator(thesecondmolecule)Theinducerisallolactose,analternativeformoflactoseNolactose,repressionVerylowleveloftranscriptionoflacZYATetramerRepressormonomerWithlactose,derepressionCataboliteRepressionofthelacOperon

乳糖操纵子的代谢物抑制

Whenglucoseispresent,lacoperonisinarelativelyinactivestateSelectioninfavorofglucoseattributedtoroleofabreakdownproduct,cataboliteProcessknownascataboliterepressionusesabreakdownproducttorepressiontheoperonPositiveControloflacOperonPositivecontroloflacoperonbyasubstancesensinglackofglucosethatrespondsbyactivatinglacpromoterTheconcentrationofnucleotide,cyclic-AMP,risesastheconcentrationofglucosedrops环腺苷酸PositiveControlofLacOperonCAPisanactivatorproteinCAPaffectspromoterCAPwilladheretopromoteronlywhenincomplexwithcAMPPositiveControl–

HighGlucoseThereislittlecAMPCAP-cAMPcomplexcannotformCAPcannotbeactivatedThepromoterisnotgoodatbindingRNApolymeraseThelactose-metabolizinggenesarenottranscribedverymuchPositiveControl–

LowGlucosecAMPaccumulatesCAP-cAMPcomplexformsComplexbindstopromoterRNApolymerasecannowbindThelactose-metabolizinggenesaretranscribedrapidlyCataboliteActivatorProtein

分解代谢激活蛋白cAMPaddedtoE.colicanecataboliterepressionoflacoperonAdditionofcAMPleadtoactivationofthelacgeneeveninthepresenceofglucosePositivecontrolleroflacoperonhas2parts:cAMPProteinfactorisknownas:CataboliteactivatorproteinorCAPCyclic-AMPreceptorproteinorCRPGeneencodingthisproteiniscrpTheMechanismofCAPAction

CAP蛋白的作用机制CAP-cAMPcomplexbindstothelacpromoterBindingofCAPandcAMPtotheactivatorsitehelpsRNApolymeraseformanopenpromotercomplexCAP-cAMP复合物结合到乳糖启动子的上游，帮助RNA聚合酶形成开放的启动子复合体ProposedCAP-cAMPActivationoflacTranscriptionTheCAP-cAMPdimerbindstoitstargetsiteontheDNATheaCTD(a-carboxyterminaldomain)ofpolymeraseinteractswithaspecificsiteonCAPBindingisstrengthenedbetweenpromoterandpolymeraseCAPbindingmakesDNAbendfor90˚CThisDNAbendingresultsinmoreefficientRNApolymerasebindingTheTrpOperonControlofGeneExpressionintheTrpOperonFeedbackcontrolNegativecontrolAttenuation

ThetrpOperonItcontainsthegenesfortheanabolicenzymesthebacteriumneedstobuildupaminoacidtryptophan含有细菌合成色氨酸需要的酶基因Anabolicenzymesaretypicallyturnedoffbyahighlevelofthesubstanceproduced当高浓度的底物存在时，合成酶被关闭。Thisoperonissubjecttonegativecontrolbyarepressorwhentryptophanlevelsareelevated当色氨酸浓度升高时，操纵子受到阻抑物的负调控。Thetrpoperonexhibitsattenuation色氨酸操纵子也表现弱化作用。ThetrpoperonLowtryptophan，norepressionWithouttryptophannotrprepressorexists,justtheinactiveprotein,aporepressor阻遏物蛋白色氨酸浓度低，无阻抑作用Hightryptophan，repression

色氨酸浓度高，有阻抑作用Ifaporepressorbindstryptophan,changesconformationwithhighaffinityfortrpoperator.Combineaporepressorandtryptophantohavethetrprepressor.Tryptophanisacorepressor共阻抑物InteractionbetweenrepressorandDNA

Attenuator弱化子162ntρ-independentterminalsequence，richofGCwith8Us，Itcanstopthetranscriptionat+140bp.LeaderRNAstructure12345‘trpEtrpL12345‘trpL234trpEtrpmRNAtrpmRNA15‘trpLtrpmRNATerminationoftranscriptionElongationoftranscriptiontrpcodonsHightrplowtrpstructuresavailabletotheleaderandattenuatorofthetrpoperonTheleaderpeptideSitesforribosomesstallwhentheleveloftrpislow.14aaDeterminetheamountoftrpandcontroltheterminationoftranscriptionFunctionoftheleader:MechanismofAttenuation

弱化作用的机制Attenuationimposesanextralevelofcontrolonanoperon,morethanjusttherepressor-operatorsystemOperatesbycausingprematureterminationoftheoperon’stranscriptwhenproductisabundantDefeatingAttenuationAttenuationoperatesintheE.colitrpoperonaslongastryptophanisplentifulIfaminoacidsupplylow,ribosomesstallatthetandemtryptophancodonsinthetrpleadertrpleaderbeingsynthesizedasstallingoccurs,stalledribosomewillinfluencethewayRNAfoldsPreventsformationofahairpin,apartofthetranscriptionterminationsignalwhichcausesattenuation.TryptophanstarvationTryptophanabundanceNoterminator,RNApol.continuesTerminatorhairpin,polymerasestopsAttenuationinthetrpOperon弱化作用Lowtryptophan,transcriptionoftrpstructure