轉(zhuǎn)錄水平的調(diào)控課件

二、轉(zhuǎn)錄水平的調(diào)控（一）原核生物的操縱子模型1.乳糖操縱子二、轉(zhuǎn)錄水平的調(diào)控（一）原核生物的操縱子模型11.乳糖操縱子大腸桿菌生長需要碳源,常見的是糖類,最方便利用的是葡萄糖,但有些條件下培養(yǎng)基中并無葡萄糖，僅有半乳糖等糖類，這時(shí)分解它們的酶類必須合成，才能利用半乳糖。1.乳糖操縱子大腸桿菌生長需要碳源,常見的是糖類,2轉(zhuǎn)錄水平的調(diào)控課件3轉(zhuǎn)錄水平的調(diào)控課件4轉(zhuǎn)錄水平的調(diào)控課件5轉(zhuǎn)錄水平的調(diào)控課件6轉(zhuǎn)錄水平的調(diào)控課件7轉(zhuǎn)錄水平的調(diào)控課件8轉(zhuǎn)錄水平的調(diào)控課件9當(dāng)葡萄糖與乳糖共存，情況比較復(fù)雜，分解它們的酶類必須不合成。降解物基因活化蛋白CAP（catabolicgeneactivateprotein,CAP),與cAMP形成復(fù)合物結(jié)合于啟動(dòng)子部位，引起DNA構(gòu)象的變化，促進(jìn)RNA聚合酶與啟動(dòng)子結(jié)合，使轉(zhuǎn)錄的起始更加頻繁，是一種正調(diào)控。當(dāng)有葡萄糖存在時(shí)，其分解代謝產(chǎn)物可抑制腺苷酸環(huán)化酶活性，激活磷酸二酯酶活性，cAMP含量下降，使CAP失活。當(dāng)葡萄糖與乳糖共存，情況比較復(fù)雜，分解它們的酶類必須不合成。10轉(zhuǎn)錄水平的調(diào)控課件11

cAMP-CAP-DNA

cAMP-CAP-DNA

12轉(zhuǎn)錄水平的調(diào)控課件13EversincethefunctionofCRPasanactivatoroftranscriptionwasestablished,agreatdealofworkhasbeendonetotryandunderstandexactlyhowthisoccurs.

EversincethefunctionofCRP14DoesCRPactivatetranscriptiondirectly?Inotherwords,doesitassistRNApolymerasetorecognizethepromoterbymeansofdirectprotein-proteincontactsbetweenCRPandRNApolymerase?

OR

DoesCRPactivatetranscriptionindirectly?SinceCRPbendsandthusdistortstheDNAdoublehelixwhenitbinds,isitpossiblethatincreasedRNApolymerasebindingisaresultofstructuralchangesintheDNA?

Athirdpossibilityisthatsomecombinationofbothdirectandindirecteffectsmightberesponsibleforactivationatindividualpromoters.DoesCRPactivatetranscriptio15Wecannowdistinguish3classesofCRP-activatedpromoter:Wecannowdistinguish3class16CLASSIClassICRP-activatedpromotersrequireonlyCRPforactivationandtheCRPbindingsiteislocatedupstreamofthepromoter.TheprototypeCLASSI

CRP-activatedpromoteristhelacoperonpromoter(lacP1).ThisCRP-bindingsiteiscentred61.5bpupstreamofthestartpointoftranscription.

CLASSI17轉(zhuǎn)錄水平的調(diào)控課件18轉(zhuǎn)錄水平的調(diào)控課件19轉(zhuǎn)錄水平的調(diào)控課件20轉(zhuǎn)錄水平的調(diào)控課件21轉(zhuǎn)錄水平的調(diào)控課件22CLASSIICLASSII

CRP-activatedpromotersrequireonlyCRPforactivationandtheCRPbindingsiteoverlapsthepromoter.Inthesepromoters,theCRPbindingsiteappearstoreplacetheusual-35regionofthepromoter.TheprototypeCLASSII

CRP-activatedpromoteristhegaloperonpromoter(galP1).ThisCRP-bindingsiteiscentred41.5bpupstreamofthestartpointoftranscription.CLASSII23CLASSIIICLASSIII

CRP-activatedpromotersrequireadditionalregulatorproteinsaswellasCRPforactivation.ThelocationoftheCRPbindingsitecanbequitevariablethoughitistypicallymorethan90bpupstreamofthestartpointoftranscription.ThereisnoprototypeCLASSIII

CRP-activatedpromotersincetheyallhavedifferentrequirementsbywayofadditionalregulatorproteins.ExamplesofCLASSIII

CRP-activatedpromotersarethearaBADpromoterandthemalKpromoterCLASSIII242.色氨酸操縱子

大腸肝菌培養(yǎng)在只含無機(jī)鹽及單一碳源的培養(yǎng)基中，大腸桿菌細(xì)胞內(nèi)可以測出色氨酸合成的酶系,如果在培養(yǎng)基中加入色氨酸,大腸桿菌中色氨酸合成的酶系就明顯降低。色氨酸存在時(shí),阻止了色氨酸合成酶系的形成,細(xì)菌可直接利用色氨酸,而不用自己合成,這種減少酶量的現(xiàn)象稱為酶合成的阻遏。2.色氨酸操縱子

大腸肝菌培養(yǎng)在只含無機(jī)鹽及單一碳源的培養(yǎng)25轉(zhuǎn)錄水平的調(diào)控課件26轉(zhuǎn)錄水平的調(diào)控課件27翻譯過程對轉(zhuǎn)錄的調(diào)節(jié)

衰減作用(弱化作用)E.Coli的色氨酸合成翻譯過程對轉(zhuǎn)錄的調(diào)節(jié)

衰減作用(弱化作用)E.Coli的色28翻譯過程對轉(zhuǎn)錄的調(diào)節(jié)

衰減作用(弱化作用)

色氨酸mRNA的5’端有162核苷酸的前導(dǎo)序列，當(dāng)RNA的合成啟動(dòng)后除非缺乏色氨酸，否則大部分mRNA僅合成140核苷酸即停止。前導(dǎo)肽能編碼一小段14肽，其終止區(qū)具有潛在的莖環(huán)構(gòu)象和成串的U，表現(xiàn)出轉(zhuǎn)錄終止位點(diǎn)的特征。前導(dǎo)RNA鏈有4個(gè)區(qū)域彼此互補(bǔ)，可形成奇特的二級結(jié)構(gòu)，有些情況下出現(xiàn)終止子結(jié)構(gòu)。翻譯過程對轉(zhuǎn)錄的調(diào)節(jié)

衰減作用(弱化作用)色氨酸mRNA的29轉(zhuǎn)錄水平的調(diào)控課件30轉(zhuǎn)錄水平的調(diào)控課件31轉(zhuǎn)錄水平的調(diào)控課件32轉(zhuǎn)錄水平的調(diào)控課件33轉(zhuǎn)錄水平的調(diào)控課件34轉(zhuǎn)錄與翻譯偶聯(lián)是原核生物的特征。原核生物翻譯結(jié)束mRNA上有特殊的終止子結(jié)構(gòu)。

轉(zhuǎn)錄與翻譯偶聯(lián)是原核生物的特征。35轉(zhuǎn)錄水平的調(diào)控課件36衰減作用(弱化作用)的意義E.Coli衰減作用(弱化作用)的意義E.Coli37E.coliBacteriumE.coliBacterium38弱化子系統(tǒng)主要是對外源色氨酸濃度作出反應(yīng)，維持濃度的恒定。色氨酸濃度t弱化子系統(tǒng)主要是對外源色氨酸濃度作出反應(yīng)，維39AUG-AAA-CGC-GUU-CAA-UUU-AAA-CAC-CAC-CAU-CAU-CAC-CAU-CAU-CCU-GACMet-Thr-Arg-Val-Gln-Phe-Lys-His-His-His-His-His-His-His-Pro-Asp-aleadercodingregion3.TheHistidineOperon:AnAttenuatorAUG-AAA-CGC-GUU-CAA-UUU-AAA-CA40轉(zhuǎn)錄水平的調(diào)控課件412．阿拉伯糖操縱子阿拉伯糖(arabinose)是另一個(gè)可以為代謝提供碳源的五碳糖。在大腸桿菌中阿拉伯糖的降解需要3個(gè)基因：araB、araA和araD，分別編碼3個(gè)酶：araB基因編碼核酮糖激酶(ribulokinase)，araA編碼L-阿拉伯糖異構(gòu)酶(L-arabinoseisomerase)，araD編碼L-核酮糖-5-磷酸-4-差向異構(gòu)酶(L-ribulose-5phosphate-4epimerase)。2．阿拉伯糖操縱子阿拉伯糖(arabinose42轉(zhuǎn)錄水平的調(diào)控課件43

與araBAD相鄰的是一個(gè)復(fù)合的啟動(dòng)子區(qū)域和一個(gè)調(diào)節(jié)基因araC，這個(gè)AraC蛋白同時(shí)顯示正、負(fù)調(diào)節(jié)因子的功能。AraBAD和araC基因的轉(zhuǎn)錄是分別在兩條鏈上以相反的方向進(jìn)行的。在標(biāo)準(zhǔn)的遺傳學(xué)圖譜上，araBAD基因簇從啟動(dòng)子PBAD開始向左進(jìn)行轉(zhuǎn)錄，而araC基因則是從Pc向右轉(zhuǎn)錄。與araBAD相鄰的是一個(gè)復(fù)合的啟動(dòng)子區(qū)域和44轉(zhuǎn)錄水平的調(diào)控課件45轉(zhuǎn)錄水平的調(diào)控課件46AraC蛋白以Crep與araO1的結(jié)合阻遏自身基因的調(diào)節(jié)并控制著自身的合成。AraC蛋白以Crep與araO1的結(jié)合阻遏自身基因的調(diào)節(jié)并47有葡萄糖無阿拉伯糖有葡萄糖無阿拉伯糖48無葡萄糖有阿拉伯糖無葡萄糖有阿拉伯糖49阿拉伯糖和葡萄糖都很低或都很高的情況下，araBCD的轉(zhuǎn)錄都被阻遏。阿拉伯糖和葡萄糖都很低或都很高的情況下，araBCD的轉(zhuǎn)錄都505.QuorumSensing:AnActivator

(群體意識）Thereexistsaspeciesofsquid（魷魚）thesquidhasevolvedalightorganinwhichitculturesaverypure,verydensepopulationofabacteriacalledVibriofischeri.Thisbacteriaproducesasubstancecalledluciferase,whichglowswiththesameintensityasthemoon.5.QuorumSensing:AnActivator51Euprymnascolopesswimming

Euprymnascolopesswimming52Euprymnascolopeshatching

Euprymnascolopeshatching53EuprymnascolopesventralviewoflightorganEuprymnascolopesventralview54Inthisventralcross-sectionofEuprymnaScolopes,thesymbioticlight-emitting"photophore"istheblackandsilver,two-lobedstructureinthecenter.CourtesyofMargaretMcFall-Ngai.bobtailsquid

Inthisventralcross-section55ThisHawaiianbobtailsquid(Euprymnascolopes)hassilverytissuearoundandacrossitseye.Thesereflective,silverytissuesoftheeyeandskinaremadeofthesameunusualproteinsthathelpmakethesquid'sflashlightwork.ThisHawaiianbobtailsquid(E56ThisHawaiianbobtailsquid(Euprymnascolopes)isanightactivepredatorthatburiesinthesandduringthedayandcomesoutatnighttoforage.Thisadultspecimen,whichispreparingtoburyfortheday,hassilverytissuesarounditseyesandblue-silvertissuesinitsskinduetoproteinscalledreflectins.ThisHawaiianbobtailsquid(E57Protein-basedreflectorsappeartobeuniquetosquid,octopusandothermembersofthecephalopodclassofmarineanimals.Mostcephalopodshavebigheads,largeeyes,grabbingtentaclesandsacsfilledwithink.

Protein-basedreflectorsappea58Close-upoftheeyeofaHawaiianbobtailsquid(Euprymnascolopes).

Close-upoftheeyeofaHawai59

WhenVibriofischeriisnotinthesquid'slightorgan,itdoesnotneedtobemakingluciferase,sinceglowingwillnothelpitoranythingelse.Ontheotherhand,wheninsidethelightorgan,itistothebacteria'sadvantagetoglow,becausethenthesquidwillnotgeteatenandwillfeedit,awayfromcompetitionfromanyotherkindsofbacteria.Sohowcanthebacteriaknowthatitisinalightorganinordertoturnonsynthesisofluciferase?WhenVibriofischeriis60TheHawaiianbobtailsquidhasabuilt-inflashlightonitsundersidewhichisbeameddownwardbystacksofsilveryreflectorplateswhicharemadefromanunusualfamilyofproteins,accordingtonewresearch.TheHawaiianbobtailsquidhas61Thesquid'slightproducingorganispoweredbyglowingbacteriawithstacksofthereflectorplatessurroundingthelightorgan.Thisglowing"flashlight"servesasaspotlightduringfeedinginthedarkocean.Thesquid'slightproducingor62轉(zhuǎn)錄水平的調(diào)控課件63Vibriofischerigrownfor24hoursonPhotobacteriumseawateragar

Vibriofischerigrownfor24h64轉(zhuǎn)錄水平的調(diào)控課件65TransmissionElectronmicrographofVibriofischeri

TransmissionElectronmicrogra66quorumsensing

EachbacteriumiscontinuouslysecretingauniquesmallmoleculecalledVAI(Vibriofischeriautoinducer)thatcandiffusereadilythroughthecellmembrane.Thus,thereisadecliningconcentrationofthesmallmoleculeinagrowingcircumferencearoundthebacterium.Whentherearemanybacteriaaround,thelocalconcentrationofthesmallmoleculewillbeveryhigh.quorumsensing

Eachbact67

Thegenesformakingluciferasearecontainedintheluxoperon.ADNAbindingsite(luxO)neartheluxpromoter(luxP)bindsaproteincalledluxR.ThisproteinsomehowcallsRNApolymeraseoverwhenitisboundtotheDNA,thusincreasingtranscriptionoftheDNAandmakingmorepolymerase.Thus,luxRisatranscriptionalactivatoroftheluxoperon.Thegenesformakinglucif68轉(zhuǎn)錄水平的調(diào)控課件69

ItisimportanttonotethatLuxIisthegenethatencodesfortheenzymethatsynthesizesVAI（autoinducer）.Whenabacteriumundergoesthetransitionfromnotmakingluciferasetomakingluciferase,itneedstohavetheautoinduceraroundinordertopromotebindingofLuxRtotheoperator.Itisimportanttonote70轉(zhuǎn)錄水平的調(diào)控課件71SerineSerine72N-acyl-L-homoserinelactones(AHLs)?；呓z氨酸內(nèi)酯N-acyl-L-homoserinelactones(73發(fā)光的的好處對于弧菌：穩(wěn)定的食物來源和生活環(huán)境。發(fā)光的的好處對于弧菌：74對于squid：誘捕消除陰影嚇阻通訊對于squid：75MargaretMcFall-Ngai,abiologistattheUniversityofHawaii.Shesaysthatwhilesymbioticrelationshipsbetweenluminescentbacteriaandanimalsarenothingnew,there'ssomeevidencethatthebacteriaaregettingarawdeal.MargaretMcFall-Ngai,abiolog76Soisthismaybelessacaseofsymbiosisthanofslavery.Thesquid,sheobserves,"inhibitsthegrowthofthebacteriatoenhancetheirluminescence."Andthebacteriumcouldmakeabetterlivingdriftingintheocean,orinthegutofanothermarineanimal,sheobserves.轉(zhuǎn)錄水平的調(diào)控課件77

細(xì)菌的許多行為,包括:生物發(fā)光、共生現(xiàn)象、生物膜形成、抗生素生產(chǎn)、群體移動(dòng)性(Swarmingmotility)、孢子形成、基因交換以及發(fā)病機(jī)理等均受到群體效應(yīng)的調(diào)節(jié)。細(xì)菌的許多行為,包括:生物發(fā)光、共生現(xiàn)象、生物78

例子銅綠假單胞菌(Pseudomonasaeruginosa)是肺囊腫性纖維化病人患慢性或致命性感染的最為常見的病原,并會感染燒燙傷或癌癥接受化療的病患。例子銅綠假單胞菌(Pseudomonas79轉(zhuǎn)錄水平的調(diào)控課件80含有銅綠假單胞菌的痰液的革蘭染色囊性纖維變性病人痰液顯示葡萄球菌和銅綠假單胞菌、細(xì)的革蘭陰性桿菌。（革蘭染色，放大1000倍）含有銅綠假單胞菌的痰液的革蘭染色囊性纖維變性病人痰液顯示葡81銅綠假單胞菌培養(yǎng)于麥康培養(yǎng)基，銅綠假單胞菌為非乳糖發(fā)酵菌，經(jīng)常產(chǎn)生稍帶綠色的綠膿菌素色素，其氧化酶陽性。（麥康基瓊脂，18小時(shí)，37℃）

銅綠假單胞菌培養(yǎng)于麥康培養(yǎng)基，銅綠假單胞菌為非乳糖發(fā)酵菌，82轉(zhuǎn)錄水平的調(diào)控課件83銅綠假單胞菌生物膜的形成受到群體效應(yīng)的調(diào)控,這種生物膜對包裹其中的銅綠假單胞菌起到一定的保護(hù)作用(避免了暴露于人體免疫反應(yīng)中,減少了與抗菌藥物的直接接觸),使得感染持久而且難以治愈。在感染早期通過基因手段切除了銅綠假單胞菌中的信息素合成酶后發(fā)現(xiàn)感染癥狀明顯減輕了。因此,自誘導(dǎo)劑的生物合成酶及自誘導(dǎo)劑感應(yīng)部位為新的抗菌藥物的開發(fā)提供了有潛力的靶點(diǎn)銅綠假單胞菌生物膜的形成受到群體效應(yīng)的調(diào)控,這種84㈢真核生物轉(zhuǎn)錄的調(diào)節(jié)㈢真核生物轉(zhuǎn)錄的調(diào)節(jié)851．轉(zhuǎn)錄因子

①順式作用成分與反式作用成分順式作用元件：轉(zhuǎn)錄其始點(diǎn)上游30bp處的TATA序列上游幾百bp的CCAAT序列或GGGCGG序列(GCbox)在基因上或下游遠(yuǎn)端或內(nèi)含子內(nèi)增強(qiáng)子(enhancer)序列負(fù)調(diào)控序列抑制子(silencer)其它特異調(diào)控序列以上各種特異的核苷酸序列都是反式作用因子蛋白質(zhì)的靶位點(diǎn)。1．轉(zhuǎn)錄因子

①順式作用成分與反式作用成分86反式作用因子：通用轉(zhuǎn)錄因子：結(jié)合在TATA附近的：TFⅡA、TFⅡB、TFⅡD、TFⅡE.轉(zhuǎn)錄調(diào)控因子：結(jié)合在上游特異核苷酸序列上的因子如SPI、CTF、AP-1、CREB還有些蛋白質(zhì)因子，它們自身并不與DNA相結(jié)合，卻能激活基因的轉(zhuǎn)錄，可能在兩種不同的調(diào)控因子中起橋梁作用。反式作用因子：872.轉(zhuǎn)錄因子的功能域(domain)

a.轉(zhuǎn)錄因子DNA結(jié)合區(qū)

Helix-turn-helix(orhelix-loop-helix)motif2.轉(zhuǎn)錄因子的功能域(domain)

a.轉(zhuǎn)錄因子D88最早在λ噬菌體CI和Cro蛋白及大腸桿菌的CAP蛋白中發(fā)現(xiàn)。雖然在氨基酸序列上有很大的可變性，但在高級結(jié)構(gòu)上是高度保守的。在兩個(gè)helix中，一個(gè)為識別螺旋，它的氨基酸殘基直接與靶DNA大溝的堿基專一結(jié)合。真核生物中最早在控制果蠅早期發(fā)育的同源域（homeodomain)蛋白中發(fā)現(xiàn)。最早在λ噬菌體CI和Cro蛋白及大腸桿菌的CAP蛋白中發(fā)現(xiàn)89CAP-cAMP

CAP-cAMP

90CAP-cAMP-DNACAP-cAMP-DNA91ZincfingermotifH2C2最早發(fā)現(xiàn)于TFIIA的DNA結(jié)合區(qū)，是RNA聚合酶轉(zhuǎn)錄5SRNA基因的必需的因子。含有堿性和極性氨基酸的的區(qū)域是識別特異DNA序列的位點(diǎn)。哺乳細(xì)胞的SP-1有類似的結(jié)構(gòu)。C4：酵母細(xì)胞的GAL-4,哺乳動(dòng)物甾體激素受體，這一類蛋白質(zhì)中的鋅指對DNA的結(jié)合是必需的，但對結(jié)合的專一性并不重要，在鋅指附近的其他氨基酸殘基與DNA直接作用形成專一結(jié)合。ZincfingermotifH2C2最早發(fā)現(xiàn)于TFII92Zincfinger(H2C2)motifZincfinger(H2C2)motif93Zincfinger(C4)motifZincfinger(C4)motif94Zincfinger(C4)Zincfinger(C4)95Zinefinger-DNAZinefinger-DNA96Zinefinger-DNAZinefinger-DNA97ZinefingerDNAZinefingerDNA98P53-DNA(exampleofzincfinger)P53-DNA(exampleofzincfinger99LeuzippermotifLeuzippermotif100酵母的GCN4，哺乳動(dòng)物的fos,jun,mys等蛋白質(zhì)DNA結(jié)合區(qū)都含有4－5個(gè)Leu，它們之間相距7個(gè)氨基酸，在helix上每兩圈出現(xiàn)一個(gè)Leu,它們排成了一行。這類蛋白質(zhì)因子都是以雙體形式與DNA靶位點(diǎn)結(jié)合，兩個(gè)蛋白質(zhì)分子相應(yīng)的helix之間靠Leu殘基的疏水作用形成一條拉鏈，對雙體的形成十分重要，但參與于DNA作用的使該區(qū)域以外的氨基酸殘基。在Leu重復(fù)區(qū)N端的鄰近的30個(gè)氨基酸片段中有許多堿性氨基酸。當(dāng)Leu拉鏈?zhǔn)箒喕纬呻p體時(shí)，堿性區(qū)處于適當(dāng)位置，決定了與DNA序列的專一結(jié)合。酵母的GCN4，哺乳動(dòng)物的fos,jun,mys等蛋白質(zhì)DN101GCN4-DNAGCN4-DNA102TrprepressorTrprepressor103β–sheetmotifβ–sheetmotif104MetrepressorMetrepressor105b.轉(zhuǎn)錄因子激活基因轉(zhuǎn)錄的功能區(qū)

酵母的GAL4,GCN4：GAL4有兩個(gè)激活轉(zhuǎn)錄的功能區(qū)，分別在147-196和768-881aa的范圍內(nèi)。GCN4的在106-125aa的肽段內(nèi)。共同特點(diǎn)是含有很多帶負(fù)電荷的α螺旋，但在aa序列上很少有同源性，aa的替換實(shí)驗(yàn)表明激活轉(zhuǎn)錄的水平與凈負(fù)電荷變化有關(guān)，增加激活區(qū)的負(fù)電荷數(shù)能提高激活轉(zhuǎn)錄的水平。哺乳動(dòng)物的AP-1、fos、jun、及糖皮質(zhì)激素受體也有類似的酸性激活區(qū)。酵母和哺乳動(dòng)物的酸性激活區(qū)可以互換。用GAL4的DNA結(jié)合區(qū)與fos的酸性激活區(qū)融合成的雜蛋白具有激活GAL基因表達(dá)的功能，同樣用fos的DNA結(jié)合區(qū)與GAL4酸性激活區(qū)融合也具有促進(jìn)基因轉(zhuǎn)錄的功能。b.轉(zhuǎn)錄因子激活基因轉(zhuǎn)錄的功能區(qū)酵母的GAL4,GCN4106SP1：結(jié)合于GCbox上，其中在N端的兩個(gè)主要結(jié)合區(qū)含有25%的谷氨酰胺。類似的有：

果蠅的Antennapedia和Zeste蛋白酵母的HAP-1、HAP-2和GAL11哺乳動(dòng)物的Oct-1、Oct-2、jun、fos、SRF等。SP1：結(jié)合于GCbox上，其中在N端的兩個(gè)主要結(jié)合區(qū)含有107CTF-1、CTF-、CTF-3和AP-2蛋白質(zhì)羧基端具有激活基因轉(zhuǎn)錄的功能，這一區(qū)域含有20-30%的脯氨酸殘基。CTF-1、CTF-、CTF-3和AP-21083.轉(zhuǎn)錄因子的作用規(guī)律

一種蛋白質(zhì)因子可以結(jié)合多個(gè)順式作用元件。一種順式作用元件可以作為多種蛋白質(zhì)因子的作用靶區(qū)。有些蛋白質(zhì)因子經(jīng)物理或化學(xué)誘導(dǎo)后才具有活性。磷酸化作用對一些蛋白質(zhì)因子的功能作用是很重要的。蛋白質(zhì)因子與特異DNA序列相互作用涉及蛋白質(zhì)因子之間的反應(yīng)。3.轉(zhuǎn)錄因子的作用規(guī)律一種蛋白質(zhì)因子可以結(jié)合多個(gè)順式作用元1094.轉(zhuǎn)錄因子與生物的性狀

同源異型突變和同源域l

指發(fā)育過程中軀體的一部分發(fā)育成另外一部分，最早在果蠅中發(fā)現(xiàn)，說明不同蛋白質(zhì)或者幾種調(diào)節(jié)蛋白質(zhì)的不同組合可以控制細(xì)胞的發(fā)育。4.轉(zhuǎn)錄因子與生物的性狀

同源異型突變和同源域l

指發(fā)育110轉(zhuǎn)錄水平的調(diào)控課件111轉(zhuǎn)錄水平的調(diào)控課件112轉(zhuǎn)錄水平的調(diào)控課件113

現(xiàn)已證明，有兩類8個(gè)同源異形基因控制胚胎最早期的空間方位，這些基因從果蠅到人類都是基本相同的。這些基因都含有180個(gè)核苷酸的序列，稱homeobox（同源域），高度保守，編碼蛋白質(zhì)羧基末端的60個(gè)氨基酸?，F(xiàn)已證明，有兩類8個(gè)同源異形基因控制胚胎最早114

這些同源異型基因編碼的都是區(qū)域特異性的轉(zhuǎn)錄因子。動(dòng)物的發(fā)育過程開始由少數(shù)幾個(gè)主基因(mastergene)控制胚胎分化的方向性，然后由不同的器官特異性基因決定器官的發(fā)生?；蚺c基因間存在明顯的等級關(guān)系。這些同源異型基因編碼的都是區(qū)域特異性的轉(zhuǎn)錄因115同源域-DNA（Helix-turn-helix）同源域-DNA（Helix-turn-helix）1165.真核生物轉(zhuǎn)錄調(diào)控的例子：

GalactoseMetabolismIsRegulatedbySpecificPositiveandNegativeControlFactorsinYeast5.真核生物轉(zhuǎn)錄調(diào)控的例子：

GalactoseMetab117MetabolismofgalactoseMetabolismofgalactose118MetabolismofgalactoseMetabolismofgalactose119Evenwhenfunctionallyrelatedgenesareclustered,theyusuallygiverisetoseparatetranscripts.Threeofthefourgenes(GAL7:transferase,GAL10:epimerase,GAL1;kinase)associatedwithgalactoseutilizationareclusteredonchromosomeXI,whereasthefourth,forgalactosetransport,isspecifiedbyagene(GAL2)locatedonchromosomeXII.Evenwhenfunctionallyrelated120Expressionofthefourstructuralgenesisbyspecificpostiveandnegativecontrols.EachofthestructuralgenesisassociatedwithadistinctmRNA.TheGAL7andGSL10genesaretranscribedfromthesameDNAstrand,whereastheGAL1gene,approximately600bpfromGAL10,istranscribedfromthecomplementaryDNAstrand.Expressionofthefourstructu121TranscriptionoftheGAL1,GAL7,andGAL10genesareincreasedover1,000-foldwhengalactoseispresent,suggestingthatgalactoseisaninducer.Twotran-actinggeneproductsthatregulateexpression:GAL4andGAL80.TranscriptionoftheGAL1,GAL7122TheactiveGAL4geneproduceisapostivecontrolprotein(anactivator)likeCAPinthelacoperon.TheGAL4proteinbindtoasiteupstreamofthegene(s)itregulatesandpromotesRNApolymeraseII-dependenttranscriptionofthesegenes.TheactiveGAL4geneproducei123SiteofbindingofGAL4proteinGAL7GAL10GAL1GAL80proteinGAL4proteinNotranscriptionNoinducerInducerpresentGAL1GAL1XISiteofbindingofGAL4protei124Mostgal80mutantsalsogiverisetoconstitutiveexpressionoftheGALgenes,whichsuggestthatGAL80normallyactsasanegativecontrolprotein(arepressor)ofGALgeneexpression.Thewild-typeGAL80proteinbindstheinducergalactoseandthatthisbindingconvertstheproteintoaninactiveform.InductionbygalacoseresultsfromthesugartotheGAL80protein,whichchangesitsstructuresothattheGAL4-GAL10complexisaltered.Mostgal80mutantsalsogiver125

酵母GAL4的激活轉(zhuǎn)錄功能區(qū)在有葡萄糖條件下被另一負(fù)調(diào)控因子GAL80結(jié)合覆蓋，失去激活轉(zhuǎn)錄的功能；經(jīng)半乳糖誘導(dǎo)，GAL4和GAL80的結(jié)合解離，GAL4的酸性激活區(qū)才促進(jìn)基因的轉(zhuǎn)錄。這是酵母半乳糖代謝酶基因在有葡萄糖條件下表達(dá)受阻遏，在有半乳糖時(shí)被誘導(dǎo)的原因。酵母GAL4的激活轉(zhuǎn)錄功能區(qū)在有葡萄糖條件下126SiteofbindingofGAL4proteinGAL7GAL10GAL1GAL80proteinGAL4proteinNotranscriptionNoinducerInducerpresentGAL1GAL1XISiteofbindingofGAL4protei127GAL4proteinisSeparatedintoDomainswithDifferentFunctionsGAL4activationdomainGAL4DNA-bindingdomainYeastGAL1geneDNARNAGAL4bindingsiteonDNAlexADNA-bindingdomainNotranscriptionlexADNA-bindingsiteonDNARNAGAL4proteinisSeparatedinto128轉(zhuǎn)錄水平的調(diào)控課件129轉(zhuǎn)錄水平的調(diào)控課件130二、轉(zhuǎn)錄水平的調(diào)控（一）原核生物的操縱子模型1.乳糖操縱子二、轉(zhuǎn)錄水平的調(diào)控（一）原核生物的操縱子模型1311.乳糖操縱子大腸桿菌生長需要碳源,常見的是糖類,最方便利用的是葡萄糖,但有些條件下培養(yǎng)基中并無葡萄糖，僅有半乳糖等糖類，這時(shí)分解它們的酶類必須合成，才能利用半乳糖。1.乳糖操縱子大腸桿菌生長需要碳源,常見的是糖類,132轉(zhuǎn)錄水平的調(diào)控課件133轉(zhuǎn)錄水平的調(diào)控課件134轉(zhuǎn)錄水平的調(diào)控課件135轉(zhuǎn)錄水平的調(diào)控課件136轉(zhuǎn)錄水平的調(diào)控課件137轉(zhuǎn)錄水平的調(diào)控課件138轉(zhuǎn)錄水平的調(diào)控課件139當(dāng)葡萄糖與乳糖共存，情況比較復(fù)雜，分解它們的酶類必須不合成。降解物基因活化蛋白CAP（catabolicgeneactivateprotein,CAP),與cAMP形成復(fù)合物結(jié)合于啟動(dòng)子部位，引起DNA構(gòu)象的變化，促進(jìn)RNA聚合酶與啟動(dòng)子結(jié)合，使轉(zhuǎn)錄的起始更加頻繁，是一種正調(diào)控。當(dāng)有葡萄糖存在時(shí)，其分解代謝產(chǎn)物可抑制腺苷酸環(huán)化酶活性，激活磷酸二酯酶活性，cAMP含量下降，使CAP失活。當(dāng)葡萄糖與乳糖共存，情況比較復(fù)雜，分解它們的酶類必須不合成。140轉(zhuǎn)錄水平的調(diào)控課件141

cAMP-CAP-DNA

cAMP-CAP-DNA

142轉(zhuǎn)錄水平的調(diào)控課件143EversincethefunctionofCRPasanactivatoroftranscriptionwasestablished,agreatdealofworkhasbeendonetotryandunderstandexactlyhowthisoccurs.

EversincethefunctionofCRP144DoesCRPactivatetranscriptiondirectly?Inotherwords,doesitassistRNApolymerasetorecognizethepromoterbymeansofdirectprotein-proteincontactsbetweenCRPandRNApolymerase?

OR

DoesCRPactivatetranscriptionindirectly?SinceCRPbendsandthusdistortstheDNAdoublehelixwhenitbinds,isitpossiblethatincreasedRNApolymerasebindingisaresultofstructuralchangesintheDNA?

Athirdpossibilityisthatsomecombinationofbothdirectandindirecteffectsmightberesponsibleforactivationatindividualpromoters.DoesCRPactivatetranscriptio145Wecannowdistinguish3classesofCRP-activatedpromoter:Wecannowdistinguish3class146CLASSIClassICRP-activatedpromotersrequireonlyCRPforactivationandtheCRPbindingsiteislocatedupstreamofthepromoter.TheprototypeCLASSI

CRP-activatedpromoteristhelacoperonpromoter(lacP1).ThisCRP-bindingsiteiscentred61.5bpupstreamofthestartpointoftranscription.

CLASSI147轉(zhuǎn)錄水平的調(diào)控課件148轉(zhuǎn)錄水平的調(diào)控課件149轉(zhuǎn)錄水平的調(diào)控課件150轉(zhuǎn)錄水平的調(diào)控課件151轉(zhuǎn)錄水平的調(diào)控課件152CLASSIICLASSII

CRP-activatedpromotersrequireonlyCRPforactivationandtheCRPbindingsiteoverlapsthepromoter.Inthesepromoters,theCRPbindingsiteappearstoreplacetheusual-35regionofthepromoter.TheprototypeCLASSII

CRP-activatedpromoteristhegaloperonpromoter(galP1).ThisCRP-bindingsiteiscentred41.5bpupstreamofthestartpointoftranscription.CLASSII153CLASSIIICLASSIII

CRP-activatedpromotersrequireadditionalregulatorproteinsaswellasCRPforactivation.ThelocationoftheCRPbindingsitecanbequitevariablethoughitistypicallymorethan90bpupstreamofthestartpointoftranscription.ThereisnoprototypeCLASSIII

CRP-activatedpromotersincetheyallhavedifferentrequirementsbywayofadditionalregulatorproteins.ExamplesofCLASSIII

CRP-activatedpromotersarethearaBADpromoterandthemalKpromoterCLASSIII1542.色氨酸操縱子

大腸肝菌培養(yǎng)在只含無機(jī)鹽及單一碳源的培養(yǎng)基中，大腸桿菌細(xì)胞內(nèi)可以測出色氨酸合成的酶系,如果在培養(yǎng)基中加入色氨酸,大腸桿菌中色氨酸合成的酶系就明顯降低。色氨酸存在時(shí),阻止了色氨酸合成酶系的形成,細(xì)菌可直接利用色氨酸,而不用自己合成,這種減少酶量的現(xiàn)象稱為酶合成的阻遏。2.色氨酸操縱子

大腸肝菌培養(yǎng)在只含無機(jī)鹽及單一碳源的培養(yǎng)155轉(zhuǎn)錄水平的調(diào)控課件156轉(zhuǎn)錄水平的調(diào)控課件157翻譯過程對轉(zhuǎn)錄的調(diào)節(jié)

衰減作用(弱化作用)E.Coli的色氨酸合成翻譯過程對轉(zhuǎn)錄的調(diào)節(jié)

衰減作用(弱化作用)E.Coli的色158翻譯過程對轉(zhuǎn)錄的調(diào)節(jié)

衰減作用(弱化作用)

色氨酸mRNA的5’端有162核苷酸的前導(dǎo)序列，當(dāng)RNA的合成啟動(dòng)后除非缺乏色氨酸，否則大部分mRNA僅合成140核苷酸即停止。前導(dǎo)肽能編碼一小段14肽，其終止區(qū)具有潛在的莖環(huán)構(gòu)象和成串的U，表現(xiàn)出轉(zhuǎn)錄終止位點(diǎn)的特征。前導(dǎo)RNA鏈有4個(gè)區(qū)域彼此互補(bǔ)，可形成奇特的二級結(jié)構(gòu)，有些情況下出現(xiàn)終止子結(jié)構(gòu)。翻譯過程對轉(zhuǎn)錄的調(diào)節(jié)

衰減作用(弱化作用)色氨酸mRNA的159轉(zhuǎn)錄水平的調(diào)控課件160轉(zhuǎn)錄水平的調(diào)控課件161轉(zhuǎn)錄水平的調(diào)控課件162轉(zhuǎn)錄水平的調(diào)控課件163轉(zhuǎn)錄水平的調(diào)控課件164轉(zhuǎn)錄與翻譯偶聯(lián)是原核生物的特征。原核生物翻譯結(jié)束mRNA上有特殊的終止子結(jié)構(gòu)。

轉(zhuǎn)錄與翻譯偶聯(lián)是原核生物的特征。165轉(zhuǎn)錄水平的調(diào)控課件166衰減作用(弱化作用)的意義E.Coli衰減作用(弱化作用)的意義E.Coli167E.coliBacteriumE.coliBacterium168弱化子系統(tǒng)主要是對外源色氨酸濃度作出反應(yīng)，維持濃度的恒定。色氨酸濃度t弱化子系統(tǒng)主要是對外源色氨酸濃度作出反應(yīng)，維169AUG-AAA-CGC-GUU-CAA-UUU-AAA-CAC-CAC-CAU-CAU-CAC-CAU-CAU-CCU-GACMet-Thr-Arg-Val-Gln-Phe-Lys-His-His-His-His-His-His-His-Pro-Asp-aleadercodingregion3.TheHistidineOperon:AnAttenuatorAUG-AAA-CGC-GUU-CAA-UUU-AAA-CA170轉(zhuǎn)錄水平的調(diào)控課件1712．阿拉伯糖操縱子阿拉伯糖(arabinose)是另一個(gè)可以為代謝提供碳源的五碳糖。在大腸桿菌中阿拉伯糖的降解需要3個(gè)基因：araB、araA和araD，分別編碼3個(gè)酶：araB基因編碼核酮糖激酶(ribulokinase)，araA編碼L-阿拉伯糖異構(gòu)酶(L-arabinoseisomerase)，araD編碼L-核酮糖-5-磷酸-4-差向異構(gòu)酶(L-ribulose-5phosphate-4epimerase)。2．阿拉伯糖操縱子阿拉伯糖(arabinose172轉(zhuǎn)錄水平的調(diào)控課件173

與araBAD相鄰的是一個(gè)復(fù)合的啟動(dòng)子區(qū)域和一個(gè)調(diào)節(jié)基因araC，這個(gè)AraC蛋白同時(shí)顯示正、負(fù)調(diào)節(jié)因子的功能。AraBAD和araC基因的轉(zhuǎn)錄是分別在兩條鏈上以相反的方向進(jìn)行的。在標(biāo)準(zhǔn)的遺傳學(xué)圖譜上，araBAD基因簇從啟動(dòng)子PBAD開始向左進(jìn)行轉(zhuǎn)錄，而araC基因則是從Pc向右轉(zhuǎn)錄。與araBAD相鄰的是一個(gè)復(fù)合的啟動(dòng)子區(qū)域和174轉(zhuǎn)錄水平的調(diào)控課件175轉(zhuǎn)錄水平的調(diào)控課件176AraC蛋白以Crep與araO1的結(jié)合阻遏自身基因的調(diào)節(jié)并控制著自身的合成。AraC蛋白以Crep與araO1的結(jié)合阻遏自身基因的調(diào)節(jié)并177有葡萄糖無阿拉伯糖有葡萄糖無阿拉伯糖178無葡萄糖有阿拉伯糖無葡萄糖有阿拉伯糖179阿拉伯糖和葡萄糖都很低或都很高的情況下，araBCD的轉(zhuǎn)錄都被阻遏。阿拉伯糖和葡萄糖都很低或都很高的情況下，araBCD的轉(zhuǎn)錄都1805.QuorumSensing:AnActivator

(群體意識）Thereexistsaspeciesofsquid（魷魚）thesquidhasevolvedalightorganinwhichitculturesaverypure,verydensepopulationofabacteriacalledVibriofischeri.Thisbacteriaproducesasubstancecalledluciferase,whichglowswiththesameintensityasthemoon.5.QuorumSensing:AnActivator181Euprymnascolopesswimming

Euprymnascolopesswimming182Euprymnascolopeshatching

Euprymnascolopeshatching183EuprymnascolopesventralviewoflightorganEuprymnascolopesventralview184Inthisventralcross-sectionofEuprymnaScolopes,thesymbioticlight-emitting"photophore"istheblackandsilver,two-lobedstructureinthecenter.CourtesyofMargaretMcFall-Ngai.bobtailsquid

Inthisventralcross-section185ThisHawaiianbobtailsquid(Euprymnascolopes)hassilverytissuearoundandacrossitseye.Thesereflective,silverytissuesoftheeyeandskinaremadeofthesameunusualproteinsthathelpmakethesquid'sflashlightwork.ThisHawaiianbobtailsquid(E186ThisHawaiianbobtailsquid(Euprymnascolopes)isanightactivepredatorthatburiesinthesandduringthedayandcomesoutatnighttoforage.Thisadultspecimen,whichispreparingtoburyfortheday,hassilverytissuesarounditseyesandblue-silvertissuesinitsskinduetoproteinscalledreflectins.ThisHawaiianbobtailsquid(E187Protein-basedreflectorsappeartobeuniquetosquid,octopusandothermembersofthecephalopodclassofmarineanimals.Mostcephalopodshavebigheads,largeeyes,grabbingtentaclesandsacsfilledwithink.

Protein-basedreflectorsappea188Close-upoftheeyeofaHawaiianbobtailsquid(Euprymnascolopes).

Close-upoftheeyeofaHawai189

WhenVibriofischeriisnotinthesquid'slightorgan,itdoesnotneedtobemakingluciferase,sinceglowingwillnothelpitoranythingelse.Ontheotherhand,wheninsidethelightorgan,itistothebacteria'sadvantagetoglow,becausethenthesquidwillnotgeteatenandwillfeedit,awayfromcompetitionfromanyotherkindsofbacteria.Sohowcanthebacteriaknowthatitisinalightorganinordertoturnonsynthesisofluciferase?WhenVibriofischeriis190TheHawaiianbobtailsquidhasabuilt-inflashlightonitsundersidewhichisbeameddownwardbystacksofsilveryreflectorplateswhicharemadefromanunusualfamilyofproteins,accordingtonewresearch.TheHawaiianbobtailsquidhas191Thesquid'slightproducingorganispoweredbyglowingbacteriawithstacksofthereflectorplatessurroundingthelightorgan.Thisglowing"flashlight"servesasaspotlightduringfeedinginthedarkocean.Thesquid'slightproducingor192轉(zhuǎn)錄水平的調(diào)控課件193Vibriofischerigrownfor24hoursonPhotobacteriumseawateragar

Vibriofischerigrownfor24h194轉(zhuǎn)錄水平的調(diào)控課件195TransmissionElectronmicrographofVibriofischeri

TransmissionElectronmicrogra196quorumsensing

EachbacteriumiscontinuouslysecretingauniquesmallmoleculecalledVAI(Vibriofischeriautoinducer)thatcandiffusereadilythroughthecellmembrane.Thus,thereisadecliningconcentrationofthesmallmoleculeinagrowingcircumferencearoundthebacterium.Whentherearemanybacteriaaround,thelocalconcentrationofthesmallmoleculewillbeveryhigh.quorumsensing

Eachbact197

Thegenesformakingluciferasearecontainedintheluxoperon.ADNAbindingsite(luxO)neartheluxpromoter(luxP)bindsaproteincalledluxR.ThisproteinsomehowcallsRNApolymeraseoverwhenitisboundtotheDNA,thusincreasingtranscriptionoftheDNAandmakingmorepolymerase.Thus,luxRisatranscriptionalactivatoroftheluxoperon.Thegenesformakinglucif198轉(zhuǎn)錄水平的調(diào)控課件199

ItisimportanttonotethatLuxIisthegenethatencodesfortheenzymethatsynthesizesVAI（autoinducer）.Whenabacteriumundergoesthetransitionfromnotmakingluciferasetomakingluciferase,itneedstohavetheautoinduceraroundinordertopromotebindingofLuxRtotheoperator.Itisimportanttonote200轉(zhuǎn)錄水平的調(diào)控課件201SerineSerine202N-acyl-L-homoserinelactones(AHLs)?；呓z氨酸內(nèi)酯N-acyl-L-homoserinelactones(203發(fā)光的的好處對于弧菌：穩(wěn)定的食物來源和生活環(huán)境。發(fā)光的的好處對于弧菌：204對于squid：誘捕消除陰影嚇阻通訊對于squid：205MargaretMcFall-Ngai,abiologistattheUniversityofHawaii.Shesaysthatwhilesymbioticrelationshipsbetweenluminescentbacteriaandanimalsarenothingnew,there'ssomeevidencethatthebacteriaaregettingarawdeal.MargaretMcFall-Ngai,abiolog206Soisthismaybelessacaseofsymbiosisthanofslavery.Thesquid,sheobserves,"inhibitsthegrowthofthebacteriatoenhancetheirluminescence."Andthebacteriumcouldmakeabetterlivingdriftingintheocean,orinthegutofanothermarineanimal,sheobserves.轉(zhuǎn)錄水平的調(diào)控課件207

細(xì)菌的許多行為,包括:生物發(fā)光、共生現(xiàn)象、生物膜形成、抗生素生產(chǎn)、群體移動(dòng)性(Swarmingmotility)、孢子形成、基因交換以及發(fā)病機(jī)理等均受到群體效應(yīng)的調(diào)節(jié)。細(xì)菌的許多行為,包括:生物發(fā)光、共生現(xiàn)象、生物208

例子銅綠假單胞菌(Pseudomonasaeruginosa)是肺囊腫性纖維化病人患慢性或致命性感染的最為常見的病原,并會感染燒燙傷或癌癥接受化療的病患。例子銅綠假單胞菌(Pseudomonas209轉(zhuǎn)錄水平的調(diào)控課件210含有銅綠假單胞菌的痰液的革蘭染色囊性纖維變性病人痰液顯示葡萄球菌和銅綠假單胞菌、細(xì)的革蘭陰性桿菌。（革蘭染色，放大1000倍）含有銅綠假單胞菌的痰液的革蘭染色囊性纖維變性病人痰液顯示葡211銅綠假單胞菌培養(yǎng)于麥康培養(yǎng)基，銅綠假單胞菌為非乳糖發(fā)酵菌，經(jīng)常產(chǎn)生稍帶綠色的綠膿菌素色素，其氧化酶陽性。（麥康基瓊脂，18小時(shí)，37℃）

銅綠假單胞菌培養(yǎng)于麥康培養(yǎng)基，銅綠假單胞菌為非乳糖發(fā)酵菌，212轉(zhuǎn)錄水平的調(diào)控課件213銅綠假單胞菌生物膜的形成受到群體效應(yīng)的調(diào)控,這種生物膜對包裹其中的銅綠假單胞菌起到一定的保護(hù)作用(避免了暴露于人體免疫反應(yīng)中,減少了與抗菌藥物的直接接觸),使得感染持久而且難以治愈。在感染早期通過基因手段切除了銅綠假單胞菌中的信息素合成酶后發(fā)現(xiàn)感染癥狀明顯減輕了。因此,自誘導(dǎo)劑的生物合成酶及自誘導(dǎo)劑感應(yīng)部位為新的抗菌藥物的開發(fā)提供了有潛力的靶點(diǎn)銅綠假單胞菌生物膜的形成受到群體效應(yīng)的調(diào)控,這種214㈢真核生物轉(zhuǎn)錄的調(diào)節(jié)㈢真核生物轉(zhuǎn)錄的調(diào)節(jié)2151．轉(zhuǎn)錄因子

①順式作用成分與反式作用成分順式作用元件：轉(zhuǎn)錄其始點(diǎn)上游30bp處的TATA序列上游幾百bp的CCAAT序列或GGGCGG序列(GCbox)在基因上或下游遠(yuǎn)端或內(nèi)含子內(nèi)增強(qiáng)子(enhancer)序列負(fù)調(diào)控序列抑制子(silencer)其它特異調(diào)控序列以上各種特異的核苷酸序列都是反式作用因子蛋白質(zhì)的靶位點(diǎn)。1．轉(zhuǎn)錄因子

①順式作用成分與反式作用成分216反式作用因子：通用轉(zhuǎn)錄因子：結(jié)合在TATA附近的：TFⅡA、TFⅡB、TFⅡD、TFⅡE.轉(zhuǎn)錄調(diào)控因子：結(jié)合在上游特異核苷酸序列上的因子如SPI、CTF、AP-1、CREB還有些蛋白質(zhì)因子，它們自身并不與DNA相結(jié)合，卻能激活基因的轉(zhuǎn)錄，可能在兩種不同的調(diào)控因子中起橋梁作用。反式作用因子：2172.轉(zhuǎn)錄因子的功能域(domain)

a.轉(zhuǎn)錄因子DNA結(jié)合區(qū)

Helix-turn-helix(orhelix-loop-helix)motif2.