2014上老師課件給學(xué)生16 sec l

SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭SectionLRegulationof

TranscriptioninProkaryotes

L1Thelacoperon

L2Thetrpoperon

L3Transcriptionalregulation

byalternativesfactor2014-04-29SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭原核基因表達(dá)調(diào)控總論基因表達(dá)調(diào)控的主要方式DNA和染色體水平調(diào)控：基因丟失、基因修飾、基因重排、基因擴(kuò)增、染色體結(jié)構(gòu)變化；轉(zhuǎn)錄水平調(diào)控（transcriptionalregulation）：轉(zhuǎn)錄的起始、延伸和終止均有影響；轉(zhuǎn)錄后水平調(diào)控（post-transcriptionalregulation）：主要指真核生物原初轉(zhuǎn)錄產(chǎn)物經(jīng)過加工成為成熟的mRNA，包括加帽、加尾、甲基化修飾等；翻譯水平調(diào)控：對(duì)mRNA穩(wěn)定性的調(diào)控、反義RNA對(duì)翻譯水平的調(diào)控等；翻譯后水平調(diào)控：蛋白質(zhì)的剪切、化學(xué)修飾（磷酸化、乙?；?、糖基化等）、轉(zhuǎn)運(yùn)等。SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭原核基因表達(dá)調(diào)控總論原核基因表達(dá)調(diào)控一般形式一個(gè)體系的基因表達(dá)通過某種“開-關(guān)”來調(diào)控；這種“開-關(guān)”主要是通過轉(zhuǎn)錄建立的，即mRNA的合成與否；所謂的“關(guān)”（off）狀態(tài)，不是絕對(duì)的，可能有本底水平的基因表達(dá)，每個(gè)世代每個(gè)細(xì)胞1-2個(gè)有mRNA分子。SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭基因轉(zhuǎn)錄調(diào)控的類型及其特點(diǎn)？正控系統(tǒng)負(fù)控系統(tǒng)調(diào)控蛋白與DNA結(jié)合基因開啟基因關(guān)閉調(diào)控蛋白與DNA分離基因關(guān)閉基因開啟SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭操縱子的類型誘導(dǎo)型操縱子在正常情況下，其基因不表達(dá)或者表達(dá)水平很低，而在環(huán)境變化或誘導(dǎo)物存在時(shí)，它才開放轉(zhuǎn)錄，生成mRNA，進(jìn)而翻譯蛋白質(zhì)，乳糖操縱子和半乳糖操縱子屬于此類（協(xié)同因子是基因表達(dá)的誘導(dǎo)物）。阻遏型操縱子操縱子是一些氨基酸合成或核苷酸合成的酶系。在培養(yǎng)基中有合成終產(chǎn)物出現(xiàn)時(shí)，會(huì)阻遏這些合成酶系的酶的合成，稱為酶的阻遏（協(xié)同因子是基因表達(dá)的共阻遏因子）。SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭細(xì)菌中的轉(zhuǎn)錄調(diào)控體系A(chǔ)負(fù)控誘導(dǎo)系統(tǒng)B正控誘導(dǎo)系統(tǒng)C負(fù)控阻遏系統(tǒng)D正控阻遏系統(tǒng)活性激活蛋白阻遏蛋白非活性阻遏蛋白誘導(dǎo)因子阻遏基因轉(zhuǎn)錄基因表達(dá)非活性激活蛋白活性激活蛋白誘導(dǎo)因子阻遏基因轉(zhuǎn)錄基因表達(dá)非活性阻遏蛋白

活性阻遏蛋白共阻遏因子共阻遏因子非活性激活蛋白阻遏基因轉(zhuǎn)錄基因表達(dá)基因表達(dá)阻遏基因轉(zhuǎn)錄SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭L1Thelacoperon(乳糖操縱子)

與負(fù)控誘導(dǎo)系統(tǒng)(A)Theoperon(操縱子)Thelactoseoperon(乳糖操縱子)Thelacrepressor(乳糖阻抑物)Induction(誘導(dǎo))cAMPreceptorprotein(cAMP受體蛋白)FrancoisJacob(44y)JacquesMonod(55y)(French)Lac.OperonTheoryNobelistsinBiologyin1965ConceptofmRNA弗朗索瓦雅各布雅克莫諾SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭Theoperon(操縱子)Definition:TheoperonisaDNAunitofgeneexpressionandregulation,whichtypicallyincludes:regulatorgene,operatorsequenceandstructuralgenes.Regulatorgenes:whoseproductsrecognizethecontrol-elements,forexamplearepressorwhichbindstoandregulatesanoperatorsequence.

Operatorsequence:Controlelementssuchasanoperatorsequence,whichisaDNAsequencethatregulatestranscriptionofthestructuralgenes.Structuralgenes:Thestructuralgenesforencodingproteins.結(jié)構(gòu)基因調(diào)節(jié)基因操縱序列PlacIlacIPlacOlaclacYlacAlacZ調(diào)控區(qū)信息區(qū)SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭Thelactoseoperon(乳糖操縱子)Structuralgenes:Thelactoseoperonconsistsof3structuralgenes:lacZ,lacY,lacA.PlacIlacIPlacOlaclacYlacAlacZmRNAb-galactosidases(b-半乳糖苷酶),permease(透性酶),Thiogalactosidetransacetylase(乙酰轉(zhuǎn)移酶).mRNAPolycistronic(多順反子)mRNA:ThethreestructuralgenesareencodedinasingletranscriptionunitlacZYA,whichhasasinglepromoterPlac,andtranscribesasinglepolycistronicmRNA,

butmoreproteinsexpressed.Lac.Operonstructureb半乳糖苷酶透性酶乙酰基轉(zhuǎn)移酶AminoacidsPolypeptideProteinStructureProductsbpmRNAPlacIPlacOlacZlacYlacA10808230691251609SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭Thelacrepressor(乳糖阻抑物)Definition:

ThelacIgeneencodestherepressor,

whichisactiveasatetramer(四聚體)ofidenticalsubunits.

Ithasaverystrongaffinityforthelacoperator-bindingsite,Olac,and

alsohasagenerallyhighaffinityforDNA.Structure:

Thelacoperator-bindingsiteconsistsof28bp

whichispalindromic（反向重復(fù)序列）.

Thisinverted(反向)symmetry(對(duì)稱)oftheoperatormatchestheinherent(內(nèi)部的)symmetryofthelacrepressor.DNASectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭Thelacrepressor(乳糖阻抑物)Binding:

Intheabsenceoflactose,therepressoroccupiestheoperator-bindingsite.

ThelacrepressorincreasesthebindingoftheRNApolymerasetothelacpromoterbytwoorderofmagnitude.(兩個(gè)數(shù)量級(jí))

ThismeansthatwhenlacrepressorisboundtotheOlac,theRNApolisalsolikelytobeboundtoadjacentPlacpromotersequencewithoutmove.SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭PlacIlacIPlacOlaclacYlacAlacZInductionprocess(誘導(dǎo))RNApLowLac.RNApRNApRNApRNApmRNA4.Transcription:5.Re-Inhibition:1.Nolactose:Intheabsenceoflactose,thelacrepressorblocksallbutaverylowleveloftranscriptionoflacZYA.2.Uptake:Whenlactoseisaddedtocell,thelowlevelofpermease(透性酶)allowsitsuptake,andb-galactosidasescatalyzessomelactosetoallo-lactose(異乳糖).3.Induction:Allo-lactoseactsasaninducer(誘導(dǎo)物)andbindstothelacrepresser.Thiscausesachangeintheconformationoftherepressortetramer,reducingitsaffinityforlacoperator.RNApRNApSectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭cAMPreceptorprotein-I(cAMP受體蛋白)Function:

?ThePlacpromoterisnotastrongpromoter.Placandrelatedpromotersdonothavestrong-35sequencesandsomeevenhaveweak-10consensussequences.Forhighleveltranscription,theyrequiretheactivityofaspecificproteincalledcAMPreceptorprotein(CRP).CRPexistsasadimerwhichcannotbindtoDNAonitsown,norregulatetranscription,butwhichcanformCRP-cAMPSectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭PositiveregulationoftheCRP（CRP的正調(diào)控）RegulatorgenePromoteroperatorlacZlacYlacACRPcAMPCRP-cAMPcomplexmRNA+CRP-cAMP(cAMPreceptorprotein環(huán)化AMP受體蛋白)bindingsite

●

CRP-cAMPbindssiteI●siteI:

IR是CRP-cAMP的強(qiáng)結(jié)合位點(diǎn)（-70~-50）●

siteII:

CRP-cAMP的弱結(jié)合位點(diǎn)（-50~-40）cooperativeeffect提高siteII

的結(jié)合效率IR-70

siteI-40siteII

IR

-50CRP-cAMP

復(fù)合體結(jié)合在SiteII(-50~-40)

促使-35附近GC島區(qū)的雙螺旋結(jié)構(gòu)穩(wěn)定性降低，使-10Sequence的解鏈溫度降低，利于轉(zhuǎn)錄啟動(dòng)

SiteI

siteIIGCIsland-35-10

●

CRP-cAMP+siteIIpromotionRNApol.into-35sequenceinto-10sequencestartingtranscriptionRNApolSectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭cAMPreceptorprotein-II(轉(zhuǎn)錄調(diào)節(jié))NormalCondition:Theglucose

(葡萄糖)

ispresentinE.coli.1.Inactivatedoperon:TheE.colidoesnotrequirealternative(替代)carbonsourcessuchaslactose.Thereforelactoseoperonisnormallyinactivated.2.InactivatedCRP:Theglucosecanreduce(降低)thelevelofcAMP,thereforethecAMP

isnotenoughforbindingCRP.SpecialCondition:Whenglucoseisabsent(缺乏)inE.coliculture.1.ActivatedCRP:ThelevelsofcAMPincreased,andCRPbindstocAMPtoformtheCRP-cAMPcomplexwhichistheactivatedCRP.2.Activatedoperon:TheCRP-cAMPcomplexbindstothePlacjustupstreamfromthesiteforRNApol.CRPbindinginducesa90obendinDNA,andtoenhanceRNApolbindingtothepromoter,enhancingtranscriptionby50-fold.SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭L2Thetrpoperon(色氨酸操縱子)

與負(fù)控阻遏系統(tǒng)(C)Thetryptophanoperon(色氨酸操縱子)Thetryrepressor(色氨酸阻抑物)Theattenuator(弱化子)LeaderRNAstructure(前導(dǎo)RNA結(jié)構(gòu))Attenuation(弱化作用)Importanceofattenuation(弱化的重要性)SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭Thetryptophanoperon(色氨酸操縱子)Structuralgenes:Thetrpoperonencodesfivestructuralgeneswhoseactivityisrequiredfortryptophansynthesis.Transcript:Theoperonencodesasingletranscriptionunitwhichproducesa7kbtranscript(多順反子)whichissynthesizeddownstreamfromthetrppromoterPtryandtrpoperatorsites(操縱基因位點(diǎn))0trp.Expression:Likemanyoftheoperonsinvolvedinaminoacidbiosynthesis,thetrpoperonhasevolved(進(jìn)化)systemsforco-ordinatedexpressionofthesegeneswhentryptophanisinshortsupplyinthecell.Regulationspeed:Aswiththe

lacoperon(如同lacoperon情況一樣),theRNAproductofthistranscriptionunitisveryunstable,enablingbacteriatorespondrapidlytochangingneedsfortryptophan.

SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭Thetrprepressor(色氨酸阻抑物)Trprepressor:AgeneproductofthetrpRoperon.Itisadimeroftwosubunits.Operatorstructure:Ptrpisbetween-21and+3.Thecorebindingsiteisapalindromeof18bp.Mechanism:

Thetrprepressorcanonlybindtotheoperatorwhenitiscomplexedwithtryptophan.

TherepressordimerhasastructurewithacentralcoreandtwoDNA-readingheads(startsite).

Whentryptophanisboundtotherepressorthereadingheadsarethecorrectdistanceapart,andthesidechainsinthecorrectconformation,tointeractwithmajorgroovesoftheDNAatPtrp.Tryptophan:istheend-productoftheenzymesencodedbythetrpoperon,itactsasaco-repressorandinhibitsitsownsynthesisbyend-productinhibition.Therepressorreducestranscriptioninitiationbyaround70-fold.trpSectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭trp與trprepressor結(jié)合改變了阻遏物的構(gòu)象SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭Theattenuator(弱化子/衰減子)Background:Atfirst,itwasthoughttherepressorwasresponsibleforallofthetranscriptionalregulationofthetrpoperon.Andthen,itwasobservedthatthedeletionofasequencebetweentheoperatorandthetrpEresultedinanincreaseinboththebasal(基本的)andtheactivated(de-repressed)levelsoftranscription.Attenuator:Thissiteistermedtheattenuatoranditliestowardstheendofthetranscribedleadersequenceof162ntthatprecedesthetrpEinitiatorcodon.Structure:Theattenuatorisa

-independentterminatorsitewhichhasashortGC-richpalindromefollowedbyeightsuccessiveUresiduesinRNAsequence.Function:Ifthissequenceisabletoforma3－4hairpinstructureintheRNAtranscript,thenitactsasahighlyefficienttranscriptionterminatorandonlya140nttranscriptissynthesized.

SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭trp弱化子位點(diǎn)堿基順序S3S4AA?UUGCAC?GG?CC?GC?GC?GG?CA?U……NNNNUUUUUUU-OHRho-independentT.

SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭Attenuation(弱化作用)Reasonofattenuation:Attenuationeffectdependsonthefactthattranscriptionandtranslationaretightlycoupled(偶聯(lián))inE.coli;translationcanoccurasanmRNAisbeingtranscribed.Sequence1:

The3’OH-endofthetrpleaderpeptidecodingsequenceoverlaps(重疊)complementarysequence1;

thetwotrpcodonsarewithinsequence1.Stopcodon:Thestopcodonintheleadersequenceisbetweensequence1andsequence2.3:4hairpin:Itisaconditionalterminator(attenuator).Whentheavailability(含量)oftryptophanisnon-starved,the3:4hairpinformsinthemRNA.

Coordination:Astranscriptionofthetrpoperonproceeds,theRNApolymerasepauses(等待)attheendofsequence2untilaribosomebeginstotranslatetheleaderpeptide.SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭Importanceofattenuation(弱化的重要性)700-fold

regulatoryeffect:Attenuation:Thepresence(存在)oftryptophangivesrisetoa10-foldrepressionoftrpoperontranscriptionthroughtheprocessofattenuationalone.Trprepressor:Combinedwithcontrolbythetrprepressor(70-fold),thismeansthattryptophanlevelsexerta700-foldregulatoryeffectonexpressionfromthetrpoperon.Hisoperon:ForexampleHistidinecodons:TheHisoperonhasaleadersequencewhichencodesapeptidewithsevensuccessivehistidinecodons.Onlymechanism:TheHisoperonhasnorepressor-operatorregulation,andattenuationformstheonlymechanismoffeedback(反饋)control.Otheroperons:Attenuationoccursinatleastsixoperonsthatencodeenzymesconcernedwithaminoacidbio-synthesis.SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭L3Transcriptionalregulationbyalternativesfactor(不同s因子對(duì)轉(zhuǎn)錄的調(diào)節(jié))Sigmafactor(s因子)Heatshock(熱休克)Bateriophagesfactor(噬菌體s因子)hsp47groelSectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭RegulationpatternsandsfactorsTranscriptionregulationpatterns:Bytranscriptionalrepressors:suchasthelacrepressor;Bytranscriptionalactivators(激活物):suchastheCRP;BydifferentstodirectRNApolbindingdifferentpromoter:Functionsofsfactors

:

Theabb’wcoreenzymeofRNApolymeraseisunabletostarttranscriptionatpromotersites.Inordertospecificallyrecognizetheconsensus-35and-10elementsofthepromoters,itrequiresthesfactorsubunit.Thissubunitisonlyrequiredfortranscriptioninitiation,beingreleasedfromthecoreenzymeafterinitiationandbeforeRNAelongationtakesplace.Features:Manybacteria,includingE.coli,produceasetofsfactorsthatrecognizedifferentsetsofpromoters.SectionL:Regulationoftranscrip.inProk.華中師范大學(xué)生科院楊旭含有

70識(shí)別位點(diǎn)的

5種大腸桿菌啟動(dòng)子TranscriptionalregulationbyalternativesfactorinE.coliforstresscondition(anexamplefortheuseofdifferents)-----When＞