真核基因表達(dá)的調(diào)控

真核基因表達(dá)的調(diào)控前言真核生物與原核生物基因表達(dá)調(diào)控的特點(diǎn)比較：

1.真核生物的基因數(shù)目比原核生物的多，而且多數(shù)基因組基因含有內(nèi)含子以及功能不清的重復(fù)序列等；原核的染色質(zhì)是裸露的DNA，而真核的染色質(zhì)則是由DNA與組蛋白緊密結(jié)合形成為核小體；在原核細(xì)胞中，染色質(zhì)的結(jié)構(gòu)對基因的表達(dá)沒有明顯的調(diào)控作用，而在真核中，這種作用是明顯的。

2.在原核基因轉(zhuǎn)錄的調(diào)控中，既有激活物的調(diào)控（正調(diào)控），也有阻遏物的調(diào)控（負(fù)調(diào)控），二者同等重要。在真核中雖然也有正調(diào)控成分和負(fù)調(diào)控成分，但迄今已知的主要是正調(diào)控。而且一個真核基因通常有多個調(diào)控序列，必須有多個激活物同時特異地結(jié)合上，才能啟動基因的轉(zhuǎn)錄；

3.原核基因的轉(zhuǎn)錄和翻譯通常偶聯(lián)在一起，而真核基因的轉(zhuǎn)錄是在細(xì)胞核中進(jìn)行，翻譯在胞質(zhì)中進(jìn)行；4.生成的初級轉(zhuǎn)錄物需在核中進(jìn)行轉(zhuǎn)錄后的加工和運(yùn)輸，所以，真核基因的表達(dá)有多種轉(zhuǎn)錄后的調(diào)控機(jī)制；

5.真核生物大都為多細(xì)胞生物，在個體發(fā)育過程中逐步分化形成各種組織和細(xì)胞類型。分化是不同基因表達(dá)的結(jié)果。不同類型的細(xì)胞，功能不同，基因表達(dá)的情況也不一樣。某些基因僅特異地在某種細(xì)胞中表達(dá)，稱為細(xì)胞特異性或組織特異性表達(dá)，因而具有調(diào)控這種特異性表達(dá)的機(jī)制。6.真核生物對外界環(huán)境條件變化的反應(yīng)和原核生物十分不同。同一群原核生物細(xì)胞處在相同的環(huán)境條件中，對環(huán)境條件的變化會作出基本一致的反應(yīng)；而真核生物常常只有少部分細(xì)胞基因的表達(dá)直接受到環(huán)境條件變化的影響和調(diào)控，其他大部分間接或不受影響。第一節(jié)染色體水平的調(diào)控一、染色體丟失某些生物，在個體發(fā)育的早期，體細(xì)胞的染色體部分丟失，而性細(xì)胞的染色體數(shù)目保持不變。例子：馬蛔蟲（Parascarisequoorum）：2n=2;

動物極植物極小表癭蚊（Mayetioledestructor）：2n=40。極細(xì)胞區(qū)：2n=402n=8二、染色體擴(kuò)增

染色體擴(kuò)增的本質(zhì)是細(xì)胞內(nèi)特定基因拷貝數(shù)的專一性大量擴(kuò)增?；蚪M序列的選擇性擴(kuò)增。以dhfr基因的擴(kuò)增為例，說明基因組序列的選擇性擴(kuò)增機(jī)制。二氫喋啶FH2FH4CoF衍生物嘌呤或嘧啶（1）合成酶；（2）還原酶。（1）（2）四氫葉酸1.類型：

StablelinesandUnstablelinesInstablelines,theamplifiedgenesareretained,becausetheyresideonthechromosome,atthesiteusuallyoccupiedbythesingledhfrgene.Usuallytheotherchromosomeretainsitsnormalsinglecopyofdhfr.

Inunstablelines,theamplifiedgenesareatleastpartiallylostwhentheselectivepressureisreleased,becausetheamplifiedgenesexistasanextrachromosomalarray.Figure17.28Thedhfrgenecanbeamplifiedtogiveunstablecopiesthatareextra-chromosomal(doubleminutes)orstable(chromosomal).Extra-chromosomalcopiesariseatearlytimes.

Figure17.29Amplifiedcopiesofthedhfrgeneproduceahomogeneouslystainingregion(HSR)inthechromosome.CHO野生型CHOrFigure17.30Amplifiedextrachromosomaldhfrgenestaketheformofdouble-minutechromosomes,asseenintheformofthesmallwhitedots.PhotographkindlyprovidedbyRobertSchimke.

2.dhfr基因擴(kuò)增的機(jī)理和特點(diǎn)

（1）機(jī)理：

非同源重組（2）特點(diǎn)：A.在用MTX加壓篩選的初期，細(xì)胞大部分或全部是不穩(wěn)定的；

B.

dhfr基因的單細(xì)胞拷貝數(shù)的變化范圍為40~400，隨著加壓程度的提高，拷貝數(shù)逐漸增加，對MTX的耐受力也相應(yīng)提高；

C.

dhfr基因的長度為31kb，但被擴(kuò)增的DNA長度可達(dá)500~1000kb；

D.當(dāng)MTX壓力解除后，dhfr基因?qū)⒅饾u減少；

E.當(dāng)與dhfr基因相連的外源DNA導(dǎo)入細(xì)胞后，有整合到內(nèi)源dhfr基因位點(diǎn)的趨勢。3.dhfr加壓系統(tǒng)的應(yīng)用EPO（促紅細(xì)胞生成素）的高效表達(dá)。pCMV/EPOCHOcellline/dhfr-StableHighExpressionMTXTransformationEPOExtraction三、染色體重排（一）酵母交配型的轉(zhuǎn)變ThematingtypeofacellisdeterminedbythegeneticinformationpresentattheMATlocus.MATaalleleatthislocusaretypea;likewise,theMATαallelearetypeα.Cellsofoppositetypescanmate;cellsofthesametypecannot.Recognitionofcellsofoppositematingtypeisaccomplishedbythesecretionof

pheromones.αcellssecreteα-factor(13aminoacids);acellssecretea-factor(12aminoacids).

Acellofonematingtypecarriesasurfacereceptorforthepheromoneoftheoppositetype.Whenanacellandanαcellencounteroneanother,theirpheromonesactoneachothertoarrestthecellsintheG1phaseofthecellcyclefollowedbycellandnuclearfusiontoproduceana/αdiploidcell.Figure17.1Matingtypecontrolsseveralactivities.

1.不同交配型的特點(diǎn)Figure17.2TheyeastlifecycleproceedsthroughmatingofMATaandMATahaploidstogiveheterozygousdiploidsthatsporulatetogeneratehaploidspores.

2.不同交配型的接合過程Figure17.3Eitheraorafactor/receptorinteractiontriggerstheactivationofaGprotein,whosebgsubunitstransducethesignaltothenextstageinthepathway.

3.交配型的信號傳遞Figure17.4Thesamematingtyperesponseistriggeredbyinteractionofeitherpheromonewithitsreceptor.Thesignalistransmittedthroughaseriesofkinasestoatranscriptionfactor;theremaybebranchestosomeofthefinalfunctions.

Figure17.5Changesofmatingtypeoccurwhensilentcassettesreplaceactivecassettesofoppositegenotype;whentranspositionsoccurbetweencassettesofthesametype,thematingtyperemainsunaltered.

4.

交配型的轉(zhuǎn)換80%~90%5%5%5.不同交配型轉(zhuǎn)換的機(jī)制WWXXXYYYa/aZ1Z1Z1Z2Z2MATa/a

HMRaHMLa/aFigure17.6Silentcassetteshavethesamesequencesasthecorrespondingactivecassettes,exceptfortheabsenceoftheextremeflankingsequencesinHMRa.OnlytheYregionchangesbetweenaandatypes.

Figure17.7Indiploidsthea1anda2proteinscooperatetorepresshaploid-specificfunctions.Inahaploids,matingfunctionsareconstitutive.Inahaploids,thea2proteinrepressesamatingfunctions,whilea1inducesamatingfunctions.

Figure17.10Cassettesubstitutionisinitiatedbyadouble-strandbreakintherecipient(MAT)locus,andmayinvolvepairingoneithersideoftheYregionwiththedonor(HMRorHML)locus.

（二）抗體的多樣性1.免疫球蛋白的產(chǎn)生、種類、結(jié)構(gòu)Theimmuneresponseofvertebratesprovidesaprotectivesystemthatdistinguishesforeignproteinsfromtheproteinsoftheorganismitself.Foreignmaterial(orpartoftheforeignmaterial)isrecognizedascomprisinganantigen.Theimmunesystemprovidesastrikingandextensivecaseinwhichthecontentofthegenomechanges,whenrecombinationcreatesactivegenesinlymphocytes.

Bcells，Tcellsmatureinthethymus.EachclassoflymphocyteusestherearrangementofDNAasamechanismforproducingtheproteinsthatenableittoparticipateintheimmuneresponse.

Forpracticalpurposes,weusuallyreckonthatamammalhastheabilitytoproduce106-108differentantibodies.

B淋巴細(xì)胞的分化過程：骨髓干細(xì)胞前B淋巴細(xì)胞未成熟B淋巴細(xì)胞成熟B淋巴細(xì)胞外周B淋巴細(xì)胞抗體骨髓外周血Figure24.1Humoralimmunityisconferredbythebindingoffreeantibodiestoantigenstoformantigen-antibodycomplexesthatareremovedfromthebloodstreambymacrophagesorthatareattackeddirectlybythecomplementproteins.

Figure24.17Immunoglobulintypeandfunctionisdeterminedbytheheavychain.JisajoiningproteininIgM;allotherIgtypesexistastetramers.

Figure24.4Heavyandlightchainscombinetogenerateanimmuno-globulinwithseveraldiscretedomains.

Eachantibodyisanimmunoglobulintetramerconsistingoftwoidenticallightchains(L)andtwoidenticalheavychains(H).Ifanylightchaincanassociatewithanyheavychain,toproduce106~108potentialantibodiesrequires103104differentlightchainsand103104differentheavychains.不同Ig家族的V、D、J、C基因片段數(shù)VDJC人小鼠人小鼠人小鼠人小鼠Lλ<3002764>64Lκ<300~10005511H~300>1000~30124498家族Figure24.5ThelambdaCgenesegmentisprecededbyaJsegment,sothatV-Jrecombinationgeneratesafunctionallambdalight-chaingene.

2.輕鏈和重鏈基因的結(jié)構(gòu)與重組Figure24.6ThekappaCgenesegmentisprecededbymultipleJsegmentsinthegermline.V-JjoiningmayrecognizeanyoneoftheJsegments,whichisthensplicedtotheCgenesegmentduringRNAprocessing.

Figure24.7Heavygenesareassembledbysequentialjoiningreactions.FirstaDsegmentisjoinedtoaJsegment;thenaVgenesegmentisjoinedtotheDsegment.

Figure24.8ThelambdafamilyconsistsofVgenesegmentslinkedtoasmallnumberofJ-Cgenesegments.

Figure24.9ThehumanandmousekappafamiliesconsistofVgenesegmentslinkedto5JsegmentsconnectedtoasingleCgenesegment.

3.抗體的多樣性Figure24.10Asinglegeneclusterinmancontainsalltheinformationforheavy-chaingeneassembly.

4.重組機(jī)制★RAG(recombinationactivegene)基因在抗體基因的重組過程中發(fā)揮了重要作用。構(gòu)成抗體多樣性的因素：輕、重鏈基因多樣性的物質(zhì)基礎(chǔ)：1）V基因的多樣性；2）D基因片段的多樣性；3）J基因片段的多樣性；4）C基因的多樣性。輕鏈V-J-C重組方式的多樣性；重鏈V-D-J-C重組方式的多樣性；輕鏈V-J和重鏈V-D連接處堿基的插入或缺失；輕鏈與重鏈的組合方式。5.抗體的表達(dá)與分泌Figure24.15AVgenepromoterisinactiveuntilrecombinationbringsitintotheproximityofanenhancerintheCgenesegment.TheenhancerisactiveonlyinBlymphocytes.第二節(jié)染色質(zhì)水平的調(diào)控一、染色質(zhì)的組成與結(jié)構(gòu)Chromatin:AmixtureiscomposedofDNA、histonesandnonhistones.Euchromatin(lowerdensity)

Heterochromatin（higherdensity）：無表達(dá)活性。Nucleosome:contains~200bpofDNA,organizedbyanoctamerofsmall,basicproteins---histonesintoabead-likestructure.Theyformaninteriorcore;theDNAliesonthesurfaceoftheparticle.Thenucleosomeprovidesthefirstleveloforganization,givingapackingratioof~6.Itscomponentsandstructurearewellcharacterized.染色體DNA

線狀，無分支。不同生物的DNA長度相差很大。生物bpDNA長度（mm）

染色體數(shù)(對)大腸桿菌4.0×1061.41酵母（S.cerevisiae）1.4×1074.616果蠅（D.malanogaster）1.7×108564人3.9×10999023Packingratio(堆積比):theratioofthelengthofDNAtotheunitlengthofthefibercontainingit.

2.組蛋白（Histones）:

H1,H2A,H2B,H3andH4

在進(jìn)化過程中，不同生物之間，同種生物的不同發(fā)育時期均高度保守，尤其H2A,H2B,H3andH4。

小牛胸腺組蛋白

組蛋白氨基酸數(shù)分子量（kDa）精（%）賴（%）H121523.0129H2A12914.0911H2B12513.8616H313515.31310H410211.31411Unitevolutionaryperiod(單位進(jìn)化周期)：兩個進(jìn)化系趨異后氨基酸序列改變1%的時間間隔。H3:3億年；H4:6億年。3.

非組蛋白（Nonhistones）Thenonhistonesincludealltheproteinsofchromatinexceptthehistones.Theyaremorevariablebetweentissuesandspecies,andtheycompriseasmallerproportionofthemassthanthehistones.Theyalsocompriseamuchlargernumberofproteins,sothatanyindividualproteinispresentinamountsmuchsmallerthananyhistone.

Thefunctionsofnonhistoneproteinsincludecontrolofgeneexpressionandhigher-orderstructure.

RNApolymerasemaybeconsideredtobeaprominentnonhistone.TheHMG(high-mobilitygroup)proteinscompriseadiscreteandwell-definedsubclassofnonhistones(atleastsomeofwhicharetranscriptionfactors).4.核小體Figure19.1Chromatinspillingoutoflysednucleiconsistsofacompactlyorganizedseriesofparticles.Thebaris100nm.Figure19.2Individualnucleosomesarereleasedbydigestionofchromatinwithmicrococcalnuclease.Thebaris100nm.Figure19.7MicrococcalnucleasedigestschromatininnucleiintoamultimericseriesofDNAbandsthatcanbeseparatedbygelelectrophoresis.Figure19.3ThenucleosomeconsistsofapproximatelyequalmassesofDNAandhistones(includingH1).Thepredictedmassofthenucleosomeis262kD.

1）組成Figure19.4ThenucleosomemaybeacylinderwithDNAorganizedintotwoturnsaroundthesurface.

2）結(jié)構(gòu)Figure19.6SequencesontheDNAthatlieondifferentturnsaroundthenucleosomemaybeclosetogether.

Figure19.10Microccocalnucleaseinitiallycleavesbetweennucleosomes.Mononucleosomestypicallyhave~200bpDNA.End-trimmingreducesthelengthofDNAfirstto~165bp,andthengeneratescoreparticleswith146bp.

Figure19.8EachmultimerofnucleosomescontainstheappropriatenumberofunitlengthsofDNA.Figure19.21Inasymmetricalmodelforthenucleosome,theH32-H42tetramerprovidesakernelfortheshape.OneH2A-H2Bdimercanbeseeninthetopview;theotherisunderneath.

3）組裝Figure19.27Invitro,DNAcaneitherinteractdirectlywithanintact(crosslinked)histoneoctamerorcanassemblewiththeH32-H42tetramer,afterwhichtwoH2A-H2Bdimersareadded.

Figure19.22Thecrystalstructureofthehistonecoreoctamerisrepresentedinaspace-fillingmodelwiththeH32-H42tetramershowninwhiteandtheH2A-H2Bdimersshowninblue.H2A-H2BH32-H42Figure19.19The10nmfiber（left）tothe30nmfiberwhichhasacoiledstructure.

5.30nm纖絲的形成Figure19.20The30nmfibermayhaveahelicalcoilof6nucleosomesperturn,organizedradially.

6.輻射狀環(huán)結(jié)構(gòu)的形成Figure18.7Histone-depletedchromosomesconsistofaproteinscaffoldtowhichloopsofDNAareanchored.

Figure18.9Thesisterchromatidsofamitoticpaireachconsistofafiber(~30nmindiameter)compactlyfoldedintothechromosome.

（一）異染色質(zhì)化（二）活潑轉(zhuǎn)錄區(qū)DNA對核酸酶的敏感性提高超敏感位點(diǎn):僅在發(fā)生基因表達(dá)的細(xì)胞中才能發(fā)現(xiàn)有這種對核酸酶超敏感的位點(diǎn)，可見它與基因的轉(zhuǎn)錄有關(guān)。已發(fā)現(xiàn)，這些位點(diǎn)通常位于被轉(zhuǎn)錄基因的5ˊ端1000bp的側(cè)翼內(nèi)，但也有位于離5ˊ端更遠(yuǎn)一些、3ˊ端和甚至基因內(nèi)部的。許多超敏感位點(diǎn)相當(dāng)于已知的調(diào)控蛋白所結(jié)合的位點(diǎn)。

（三）正在轉(zhuǎn)錄的染色質(zhì)處DNA的甲基化程度降低（四）正在轉(zhuǎn)錄區(qū)的組蛋白和有關(guān)蛋白質(zhì)發(fā)生改變

活潑轉(zhuǎn)錄的染色質(zhì)趨于缺乏組蛋白H1，而且其它的核心組蛋白則被乙酰基化或與泛素（ubiquitin）相結(jié)合而被修飾。

染色質(zhì)的體外重組實驗二、染色質(zhì)的結(jié)構(gòu)對基因表達(dá)的調(diào)控染色質(zhì)體外重組實驗：

用鹽或稀酸處理染色質(zhì)，可將DNA、組蛋白和非組蛋白分開，經(jīng)離子交換層析、乙醇沉淀等技術(shù)處理可分別獲得上述三種組分。來自不同細(xì)胞的染色質(zhì)組分混合后，能夠重新組建成染色質(zhì)。

將無轉(zhuǎn)錄活性細(xì)胞的DNA和組蛋白與有轉(zhuǎn)錄活性細(xì)胞的非組蛋白混合，則產(chǎn)生轉(zhuǎn)錄活性。第三節(jié)轉(zhuǎn)錄和轉(zhuǎn)錄后加工的調(diào)控一、順式作用元件與反式作用因子（一）順式作用元件（Cis-actingsequences）啟動子核心成分：如TATAbox；上游啟動子成分：如CAATbox，GCbox、八聚體（octamer）以及ATF結(jié)合位點(diǎn)等；遠(yuǎn)上（下）游序列：如增強(qiáng)子，酵母的UAS（upstreamactivatorsequences），靜息子等；特殊細(xì)胞中的啟動子成分：淋巴細(xì)胞中的Oct和κB等。哺乳動物RNApolymerase啟動子上游轉(zhuǎn)錄因子結(jié)合的序列元件組件保守序列DNA長度結(jié)合因子大小（kDa）豐度/細(xì)胞分布TATAboxTATAAA-10bpTBP27?普遍CAATboxGGCCAATCT-22bpCTF/NF160300000普遍GCboxGGGCGG-20bpSP116560000普遍OctamerATTTGCAT-20bpOct-176?普遍OctamerATTTGCAT23bpOct-252?淋巴細(xì)胞κBGGGACTTTCG-10bpNFκB44?淋巴細(xì)胞κBGGGACTTTCG-10bpH2.TH1??普遍ATFGTGACGT-20bpATF??普遍結(jié)合于反應(yīng)性元件的特殊轉(zhuǎn)錄因子調(diào)節(jié)劑反應(yīng)元件保守序列長度反應(yīng)因子大小（kDa）熱休克HSECNNGAANNTCCNNG27bpHSTF93糖皮質(zhì)激素GRETGGTACAAATGTTCT20bp受體94佛波酯TRETGACTCA22bpAP139血清SRECCATATTAGG20bpSRF52

（二）反式作用因子(Trans-actingelement)通用反式作用因子：一般細(xì)胞中普遍存在，如TBP，SP1，CTF/NF-1，Oct-1等;特殊組織和細(xì)胞中的反式作用因子：Oct-2;與反應(yīng)性元件結(jié)合的反式作用因子:見下表。反式作用因子的調(diào)控作用途徑：1）蛋白質(zhì)與DNA相互作用；2）蛋白質(zhì)之間的相互作用；3）蛋白質(zhì)與配基結(jié)合；4）蛋白質(zhì)自身的修飾。Figure21.2Theactivityofaregulatorytranscriptionfactormaybecontrolledbysynthesisofprotein,covalentmodificationofprotein,ligandbinding,orbindingofinhibitorsthatsequestertheproteinoraffectitsabilitytobindtoDNA.

二、調(diào)控蛋白與DNA的結(jié)合方式調(diào)控蛋白通常是結(jié)合在特異的DNA序列上而發(fā)揮作用的。而且調(diào)控蛋白通常有獨(dú)立的結(jié)合DNA的結(jié)構(gòu)域。（一）螺旋－轉(zhuǎn)角－螺旋（helix-turn-helix）（二）鋅指（ZincFinger）（三）同源異型結(jié)構(gòu)域（homeodomains,HD）Figure10.13ThestructureofamonomerofLacrepressoridentifiesseveralindependentdomains.（一）螺旋－轉(zhuǎn)角－螺旋（helix-turn-helix

）Thehelix-turn-helixmotifwasoriginallyidentifiedastheDNA-bindingdomainofphagerepressors.Oneα-helixliesinthewidegrooveofDNA;theotherliesatanangleacrossDNA.Figure10.15InducerchangesthestructureofthecoresothattheheadpiecesofarepressordimerarenolongerinanorientationthatpermitsbindingtoDNA.Figure21.12Helix3ofthehomeodomainbindsinthemajorgrooveofDNA,withhelices1and2lyingoutsidethedoublehelix.Helix3contactsboththephosphatebackboneandspecificbases.TheN-terminalarmliesintheminorgroove,andmakesadditionalcontacts.

（二）鋅指（ZincFinger）

ThezincfingermotifcomprisesaDNA-bindingdomain.ItwasoriginallyrecognizedinfactorTFIIIA,whichisrequiredforRNApolymeraseIIItotranscribe5SrRNAgenes.Ithassincebeenidentifiedinseveralothertranscriptionfactors(andpresumedtranscriptionfactors).AdistinctformofthemotifisfoundalsointhesteroidreceptorsThesteroidreceptorsaredefinedasagroupbyafunctionalrelationship:eachreceptorisactivatedbybindingaparticularsteroid.Theglucocorticoidreceptoristhemostfullyanalyzed.Togetherwithotherreceptors,suchasthethyroidhormonereceptorortheretinoicacidreceptor,thesteroidreceptorsaremembersofasuperfamilyoftranscriptionfactorswiththesamegeneralmodusoperandi.

Figure21.3TranscriptionfactorSP1hasaseriesofthreezincfingers,eachwithacharacteristicpatternofcysteineandhistidineresiduesthatconstitutethezinc-bindingsite.Figure21.4Zincfingersmayforma-helicesthatinsertintothemajorgroove,associatedwithb-sheetsontheotherside.

Figure21.5ThefirstfingerofasteroidreceptorcontrolsspecificityofDNA-binding(positionsshowninred);thesecondfingercontrolsspecificityofdimerization(positionsshowninblue).TheexpandedviewofthefirstfingershowsthatdiscriminationbetweenGREandEREtargetsequencesrestsontwoaminoacidsatthebase.

三、調(diào)節(jié)蛋白與蛋白質(zhì)結(jié)合的方式在真核細(xì)胞中，許多轉(zhuǎn)錄因子以二聚體結(jié)合在DNA上。這些調(diào)控蛋白除了與DNA結(jié)合的結(jié)構(gòu)外，還有與蛋白質(zhì)結(jié)合的結(jié)構(gòu)域。這些結(jié)構(gòu)域參與二聚體的形成。由相同亞基組成的二聚體，稱為同二聚體（homodimer）;由不同亞基所組成的二聚體，稱為異二聚體（heterodimer）。二聚體的形成是它與DNA結(jié)合的必要條件。和蛋白質(zhì)與DNA結(jié)合的基元一樣，媒介蛋白質(zhì)－蛋白質(zhì)相互作用的蛋白質(zhì)結(jié)構(gòu)基元可歸屬于幾種類型。其中鑒定得比較清楚的有螺旋－環(huán)－螺旋和亮氨酸拉鏈兩種。

(一)螺旋－環(huán)－螺旋（helix-loop-helix,HLH）

(二)亮氨酸拉鏈（Leucinezippers）（一）螺旋－環(huán)－螺旋（helix-loop-helix，HLH）Theamphipathichelix-loop-helix(HLH)motifhasbeenidentifiedinsomedevelopmentalregulatorsandingenescodingforeukaryoticDNA-bindingproteins.Eachamphipathichelixpresentsafaceofhydrophobicresiduesononesideandchargedresiduesontheotherside.Thelengthoftheconnectingloopvariesfrom12~28aminoacids.Themotifenablesproteinstodimerize,andabasicregionnearthismotifcontactsDNA.

Figure21.13AllHLHproteinshaveregionscorrespondingtohelix1andhelix2,separatedbyaloopof10-24residues.BasicHLHproteinshavearegionwithconservedpositivechargesimmediatelyadjacenttohelix1.

Figure21.14AnHLHdimerinwhichbothsubunitsareofthebHLHtypecanbindDNA,butadimerinwhichonesubunitlacksthebasicregioncannotbindDNA.

（二）亮氨酸拉鏈（Leucinezippers）Leucinezippersconsistofastretchofaminoacidswithaleucineresidueineveryseventhposition.Aleucinezipperinonepolypeptideinteractswithazipperinanotherpolypeptidetoformadimer.AdjacenttoeachzipperisastretchofpositivelychargedresiduesthatisinvolvedinbindingtoDNA.Figure21.15ThebasicregionsofthebZIPmotifareheldtogetherbythedimerizationattheadjacentzipperregionwhenthehydrophobicfacesoftwoleucinezippersinteractinparallelorientation.四、轉(zhuǎn)錄起始與轉(zhuǎn)錄后加工的調(diào)控

轉(zhuǎn)錄起始與轉(zhuǎn)錄后加工的調(diào)控主要包括以下幾個方面的內(nèi)容：

轉(zhuǎn)錄起始的選擇；mRNA前體的可變剪接(alternativesplicing)；RNA編輯（RNAediting）；

mRNA的運(yùn)輸和穩(wěn)定性的調(diào)控。（一）mRNA前體的剪接機(jī)制mRNA前體的加工包括5ˊ加帽、3ˊ加尾、剪接和編輯等。含有內(nèi)含子的轉(zhuǎn)錄物中剪接區(qū)的堿基序列基因區(qū)域外顯子內(nèi)含子外顯子卵清蛋白內(nèi)含子2UAAGGUGAGC---------UUACAGGUUG卵清蛋白內(nèi)含子3UCAGGUACAG---------AUUCAGUCUGβ‐珠蛋白內(nèi)含子1GCAGGUUGGU---------CCUUAGGCUGβ‐珠蛋白內(nèi)含子2CAGGGUGAGU―――CCACAGUCUC免疫蛋白λ內(nèi)含子1UCAGGUCAGC―――UUGACGGGGCSV40病毒早期T-抗原UAAGGUAAAU―――UUUUAGAUUC1.真核生物基因內(nèi)含子的結(jié)構(gòu)特點(diǎn)Figure22.3Theendsofnuclearintronsaredefinedbythe

GT-AGrule.真核生物基因內(nèi)含子的結(jié)構(gòu)特點(diǎn)：1.內(nèi)含子的大小:50～10000nt；2.5’-末端絕大多數(shù)為GU開始；3’-末端絕大多數(shù)為AG結(jié)束(GU-ATrule)；3.在3’-末端的上游20～50nt處，有一段稱為分支點(diǎn)（branchsite）的序列，在酵母中，此序列幾乎都是UACUAAC，哺乳動物中有多種不同的序列；4.在5’-末端、3’-末端及分支點(diǎn)保持不變的情況下，可在內(nèi)含子中插入或刪除不同大小的外源基因片段，而且不影響基因的加工。Figure22.2RNAismodifiedinthenucleusbyadditionstothe5’and3’endsandbysplicingtoremovetheintrons.Thesplicingeventrequiresbreakageoftheexon-intronjunctionsandjoiningoftheendsoftheexons;MaturemRNAistransportedthroughnuclearporestothecytoplasm,whereitistranslated.

２.剪接體催化的剪接機(jī)制（1）剪接體的定義在細(xì)胞核和胞質(zhì)溶膠中都含有多種不到300個核苷酸的小RNA分子，它們被稱為核內(nèi)小RNA（smallnuclearRNA，snRNA）和胞質(zhì)內(nèi)小RNA(smallcytoplasmicRNA，scRNA)。這些RNA分子和特異的蛋白質(zhì)結(jié)合在一起形成復(fù)合體，這些復(fù)合體分別叫做核內(nèi)小核糖核蛋白顆粒（smallnuclearribonucleoproteinparticles，snRNPs）和胞質(zhì)內(nèi)小核糖核蛋白顆粒（smallcytosolicribonucleopeinparticles，scRNPs）。研究者們也常常把它們叫做“snurps”和“scurps”。

剪接體（spliceosome）是一類大的復(fù)合體(60s)，它們是由mRNA前體和snRNPs動態(tài)的聚集在一起而形成的。參與mRNA前體剪接的snRNPs

snRNPsnRNA大?。╪t）作用U1165先后結(jié)合在5’、3’剪接位點(diǎn)上，U2185與分支位點(diǎn)結(jié)合，形成催化中心的一部分U5116結(jié)合在5’剪接位點(diǎn)上U4145遮蔽U6的催化活性U6106催化剪接Figure22.10Thesplicingreactionproceedsthroughdiscretestagesinwhichspliceosomeformationinvolvestheinteractionofcomponentsthatrecognizetheconsensussequences.

（2）剪接體的組裝過程Figure22.14Spliceosomesareellipsoidalparticleswithseveraldiscreteregions.Thebaris50nm.

Figure22.6Splicingoccursintwostages,inwhichthe5’-exonisseparatedandthenisjoinedtothe3’-exon.

（3）剪接體催化的剪接過程Figure22.7NuclearsplicingoccursbytwotransesterificationreactionsinwhichafreeOHendattacksaphosphodiesterbond.Figure22.8U1snRNAhasabasepairedstructurethatcreatesseveraldomains.The5’-endremainssinglestrandedandcanbasepairwiththe5’-splicingsite.

Figure22.12U6-U4pairingisincompatiblewithU6-U2pairing.WhenU6joinsthespliceosomeitispairedwithU4.ReleaseofU4allowsaconformationalchangeinU6;onepartofthereleasedsequenceformsahairpin(darkgrey),andtheotherpart(black)pairswithU2.BecauseanadjacentregionofU2isalreadypairedwiththebranchsite,thisbringsU6intojuxtapositionwiththebranch.NotethatthesubstrateRNAisreversedfromtheusualorientationandisshown3’–5’（二）可變剪接1.可變剪接的方式現(xiàn)在已知許多基因有兩個或更多個啟動子，因而有兩個或更多個轉(zhuǎn)錄起始位點(diǎn)。

（1）選用不同的啟動子當(dāng)用不同的啟動子進(jìn)行轉(zhuǎn)錄時，產(chǎn)生的初始轉(zhuǎn)錄物是不同的。如肌紅蛋白基因。

（2）選用不同的加尾位點(diǎn)許多基因的初始轉(zhuǎn)錄物有多個加尾位點(diǎn)。選用不同的加尾位點(diǎn)可產(chǎn)生出不同的蛋白質(zhì)。

（3）選用不同的剪接位點(diǎn)如SV40的T和t抗原是由同一基因用這種方式產(chǎn)生出來的兩種蛋白質(zhì)。

（4）上述二者兼有：如降鈣素基因轉(zhuǎn)錄產(chǎn)物的剪接方式。Figure22.4Splicingjunctionsarerecognizedonlyinthecorrectpairwisecombinations.

Figure22.5NorthernblottingofnuclearRNAwithanovomucoidprobeidentifiesdiscreteprecursorstomRNA.Thecontentsofthemoreprominentbandsareindicated.Figure22.18Alternativeformsofsplicingmaygenerateavarietyofproteinproductsfromanindividualgene.Changingthesplicesitesmayintroduceterminationcodons(shownbyasterisks)orchangereadingframes

Figure22.20Alternativesplicingeventsthatinvolvebothsitesmaycauseexonstobeaddedorsubstituted.

2.mRNA前體可變剪接的概念和意義大多數(shù)真核基因轉(zhuǎn)錄產(chǎn)生的mRNA前體是按一種方式剪接產(chǎn)生出一種mRNA，因而只產(chǎn)生一種蛋白質(zhì)。但有些基因產(chǎn)生的mRNA前體可按不同的方式剪接，產(chǎn)生出兩種或更多種mRNA，即可變剪接（alternativesplicing）。為什么真核基因含有那么多內(nèi)含子？迄今仍不甚明了。但可變剪接可以說明內(nèi)含子的功能之一，即由于內(nèi)含子的存在使得可變剪接成為可能。其意義在于可使一個基因表達(dá)出多種蛋白質(zhì)，即擴(kuò)大了DNA中遺傳信息的含量。然而令人不解的是，高等真核生物的基因組是很大的，它完全能夠容納更多的基因，但不知為什么一方面它的許多基因非常分散，另一方面它又使一個基因產(chǎn)生出多種產(chǎn)物。（三）RNA編輯（RNAediting）

RNAeditingisaprocessinwhichinformationchangesatthelevelofmRN