第十一章 核酸的合成2

DNAandRNAsynthesesaresimilarinsomeaspectsbutdifferentinothersBothreplicationandtranscription(RNAsynthesis)areguidedbyatemplateviaWatson-Crickbasepairing;Bothhavepolarity,proceedingin5’3’direction;Bothusetriphosphatenucleotides(dNTPorNTP);But:

NoprimersareneededforRNAsynthesis(transcription);RNAsynthesisonlyinvolvesashortsegmentofalargeDNAmolecule;RNAsynthesisusesonlyoneofthetwoDNAstrandsastemplateatonesite.

在起始合成前，RNA聚合酶先與DNA模板鏈相結(jié)合，只有一條鏈作為模板進(jìn)行RNA的合成，故稱為不對(duì)稱轉(zhuǎn)錄，這條轉(zhuǎn)錄RNA的模板DNA鏈稱為模板鏈或反義鏈，另一條稱為有義鏈。不對(duì)稱轉(zhuǎn)錄有義鏈和反義鏈時(shí)間有義鏈反義鏈1990年前模板鏈非模板鏈1990年以后非模板鏈模板鏈第二節(jié)RNA的生物合成

一、轉(zhuǎn)錄在DNA指導(dǎo)下的RNA合成稱為轉(zhuǎn)錄。轉(zhuǎn)錄從DNA模板的一個(gè)特定位點(diǎn)開(kāi)始，到另一位點(diǎn)終止。此轉(zhuǎn)錄區(qū)域稱為轉(zhuǎn)錄單位，一個(gè)轉(zhuǎn)錄單位可以是一個(gè)基因，也可以是多個(gè)基因，DNA的啟動(dòng)子控制轉(zhuǎn)錄的起始，而終止子控制轉(zhuǎn)錄的終止。n1ATPn2GTPRNA聚合酶n3UTP---------------→RNA+(n1+n2+n3+n4)ppin4CTPDNA模板，Mg2+轉(zhuǎn)錄是由DNA指導(dǎo)下的RNA聚合酶催化進(jìn)行的，反應(yīng)如下:

MW,46萬(wàn),五個(gè)亞基兩個(gè)α亞基，一個(gè)β亞基,一個(gè)β’亞基和σ因子，含有2個(gè)Zn2+離子，與β’相連接。沒(méi)有σ亞基時(shí)稱為核心酶，核心酶有催化聚合反應(yīng)的活性，σ為起始因子，識(shí)別起始位點(diǎn)。全酶制劑中還存在一種分子量較小的ω亞基，核心酶沒(méi)有。1.

原核生物RNA聚合酶TheE.coliRNApolymeraseconsistsoffourtypesofsubunitsHoloenzyme:a2bb’

sCoreenzyme:

a2bb’

OneRNApolymeraseisusedforsynthesizingall

theRNAmoleculesinbacteria!TherearethreekindsofRNApolymerases(I,II,andIII)ineukaryoticcellsThenucleusofeukaryoticcellscontainsthreetypesofRNApolymerasesdifferingintemplatespecificity,localization,andsusceptibilitytoa-amanitin;RNApolymeraseIIbindstopromotersofthousandsofprotein-codinggeneswiththeaidofvarioustranscriptionfactorstoproducethethousandsofmRNAmolecules.EukaryoticRNApolymerasesa-amanitin：何方神圣？中文名：鵝膏蕈堿存在于毒麻菇Amanitaphalloides的毒素Amanitaphalloidesa-amanitin環(huán)八肽2GlyL-IleL-二羥IleL-AsnL-羥ProL-羥Trp氧化Cys2.RNA的合成過(guò)程:(分為4步)(1)RNA聚合酶與DNA模板的結(jié)合RNA聚合酶在有義鏈的啟動(dòng)子部位結(jié)合。啟動(dòng)子:指RNA聚合酶識(shí)別、結(jié)合和開(kāi)始轉(zhuǎn)錄的一段DNA序列，σ識(shí)別啟動(dòng)部位。這一部位富含AT，熔點(diǎn)(Tm)較低，雙鏈容易打開(kāi)。RNA聚合酶在此與DNA形成復(fù)合物,并沿模板3’→5’方向移動(dòng)。

promotertwoconsensussequences;At-10:TATAAT(alsocalledthePribnowbox,TATAbox);At-35:E.coli46個(gè)啟動(dòng)子(Schaller和Gilbert):-35區(qū)-10區(qū)········T·T·G·A·C·A·············T·A·T·A·A·T·G··············

38373527292840412529304617-10sequences(Pribnowbox)-35sequences+1ThessubunitsenabletheE.coliRNApolymerasetorecognizepromotersitesTheRNApolymerasewithoutthessubunit(i.e.,thea2bb’)isunabletostarttranscriptionatpromotersites;ThessubunitdecreasetheaffinityofRNApolymeraseforgeneral(non-promoter)regionsofDNAbyafactorof104;E.colicontainsmultiplesfactorsforrecognizingdifferentpromoters:s70forstandardpromoters;s32forheat-shockpromoters;s54

fornitrogen-starvationpromoters;Eachtypeofsfactorallowsthecelltocoordinatelyexpressasetofgenes.StandardHeat-shockNitrogenstarvations70forstandardpromoters;

s32forheat-shockpromoters;

s54fornitrogen-starvationpromotersE.colicontainsmultiplesfactorsforrecognizingdifferenttypesofpromoters:(2)轉(zhuǎn)錄起始全酶中β亞基催化RNA的第一個(gè)核苷酸(一般是ATP和GTP)的磷酸二酯鍵的形成，一旦ATPorGTP接上去后，σ因子便脫離下來(lái)，這時(shí)降低酶對(duì)啟動(dòng)子的親和力，剩下的核心酶與DNA結(jié)合松馳，有利于核心酶沿模板鏈移動(dòng)，RNA鏈的延長(zhǎng)，σ又與另一分子的核心酶結(jié)合，起動(dòng)一個(gè)新的RNA鏈的合成。ThesfactorhelpsinitiateRNAsynthesisatpromoterssss(3)鏈的延長(zhǎng):模板方向3’→5’，RNA5’→3’，這個(gè)反應(yīng)的△G負(fù)值很大，使RNA鏈延長(zhǎng)較快，正在轉(zhuǎn)錄的區(qū)域，DNA雙鏈解開(kāi)使新進(jìn)入核苷酸與DNA鏈配對(duì)，已轉(zhuǎn)錄完的DNA鏈則重新形成雙螺旋。(4)轉(zhuǎn)錄的終止提供轉(zhuǎn)錄停止信號(hào)的DNA序列稱為終止子(terminator)，幫助RNA聚合酶識(shí)別終止信號(hào)的輔助因子則稱為終止因子，如ρ因子。當(dāng)RNA聚合酶沿模板鏈移動(dòng)到一個(gè)基因的末端時(shí)，基因末端給予一個(gè)終止信號(hào)，使轉(zhuǎn)錄終止。ThelengthofRNAmoleculessynthesizedinvitrovarywithandwithoutaddingtherprotein.M13phageDNAAr-independentterminator:amodelPalindromeDNAsequencesStem-loop(hairpin)structureρ因子20萬(wàn)(分子量)六個(gè)亞基，專一識(shí)別終止子，阻止RNA聚合酶向前移動(dòng)，于是轉(zhuǎn)錄終止，釋放RNA鏈。

原核生物轉(zhuǎn)錄真核生物轉(zhuǎn)錄TheactionofRNApolymerasescanbespecificallysuppressedThree-ring-containing,planarantibioticmoleculeslikeactinomycinDintercalatesbetweentwosuccessiveGCbasepairsinduplexDNA,preventingRNApolymerasetomovealongthetemplate(thustheelongationofRNAsynthesis);Rifampicin(anantibiotic)bindstothebsubunitofbacterialRNApolymerases,preventingthe

initiationofRNAsynthesis;a-amanitin

轉(zhuǎn)錄的RNA原初產(chǎn)物需要經(jīng)過(guò)一系列的拼接、裂解、修飾才具有生物功能,這個(gè)過(guò)程稱為加工。

4.RNA轉(zhuǎn)錄后的加工

原核生物mRNA一般不需要加工，一經(jīng)轉(zhuǎn)錄，即可翻譯，真核生物轉(zhuǎn)錄的mRNA含有內(nèi)含子和外顯子的分子很大，很不均一，稱hnRNA，(1)真核細(xì)胞mRNA的加工AneukaryoticmRNAprecursoracquirea5`capshortlyaftertranscriptioninitiatesAGMPcomponentisjoinedtothe5`endofthemRNAinanovel5`,5`-triphosphatelinkage;TheguaninebaseisthenmethylatedattheN-7;The2`-OHgroupsofthe1stand2ndnucleotidesadjacenttothe7-methylguaninecapmayalsobemethylatedincertainorganisms;ThemethylgroupsaretransferredfromS-adenosylhomocysteine.The5`capofmRNAanditsformationprocessMosteukaryoticmRNAs(excepthistonemRNAs)haveapoly(A)tailatthe3`endThetailconsistsof20to250adenylateresidues;ThemRNAprecursorsareextendedbeyondthesitewherepoly(A)tailistobeadded;CleavagesignalAnAAUAAAsequencewasfoundtobepresentinallmRNAsmarking(togetherwithothersignalsatthe3`end)thesiteforcleavageandpoly(A)tailaddition(11to30nucleotidesonthe3`endoftheAAUAAAsequence);Thespecificendonucleaseandpolyadenylatepolymerase,andotherproteinsprobablyexistasamultiproteincomplextoperformtheprocessing.RNAsplicingSinglestrandedDNAloopswereobservedwhenmaturechickenovalbuminmRNAwashybridizedwithdenaturedDNAcontainingtheovalbumingene(R-loopingtechnique).DNARNAeditingCertainmRNAswerefoundtobeeditedpriortotranslation

Thesephenomenawasdiscoveredinthemid-1980s;Deaminationofacytosineresidueoftheapo-BmRNAinmammalianintestineconvertsaGln-codingCAAcondontotheUAAstopcodon;ManymRNAmoleculesinthemitochondriaoftrypanosomeshavebeenfoundtobeextensivelyeditedbytheinsertionanddeletionofUresidues;TheextensiveRNAeditingintrypanosomesisdirectedbysmallguideRNAs(gRNAs).錐體蟲(chóng)線粒體細(xì)胞色素C氧化酶亞基Ⅱ(CoⅡ)基因

480490500510

mRNA：UUAGGUAUAAAAGUAGAUUGUAUACCUGGUAGGUGUAAU蛋白序列

：L

G

I

K

V

D

C

I

P

G

R

C

N

DNA：TTAGGTATAAAAGTAGA**G*A*ACCTGGT

AGGTGTAAT核酸序號(hào)以mRNA起始密碼AUG的A開(kāi)始編號(hào)。下劃線標(biāo)出與人及酵母相應(yīng)蛋白相同的氨基酸。錐體蟲(chóng)T.buucei亞種mRNA的編輯(不是全部)mRNA起始密碼

編輯區(qū)域5’區(qū)

編碼區(qū)3’及PolyA區(qū)CoⅡ+／－4／0CoШ－／？394／1832／1Cyb

+／+34／014／0MURF2－／+26／4

斜線坐標(biāo)時(shí)插右邊表示刪去U。斜線右表示基因中有無(wú)起始密碼。右邊表示編輯中有無(wú)構(gòu)建起始密碼。TheextensiveRNAeditingintrypanosomesisdirectedbysmallguideRNAs(gRNAs).(2)rRNA前體的加工原核生物與真核生物的rRNA前體都較長(zhǎng),大腸肝菌中加工過(guò)程:

↗17.5S→16SrRNA+核糖體Pr小亞基30S原核生物30S→25S→23SrRNA+核糖體Pr大亞基50S↘小碎片→5SrRNA

↗18S40S小亞基45S甲基化→28S80S內(nèi)切↘5.8S60S大亞基其它途徑產(chǎn)生→5SProcessingofbacterialpre-rRNAProcessingofeukaryoticpre-rRNA(2)tRNA前體的加工數(shù)十種tRNA加工不同,大致如下:①切除5’末端,3’末端多余部分②在3’末端加上CCA-OH序列③核苷的修飾:甲基化、尿嘧啶移位,假尿嘧啶的生成DHUIntron(groupIV)PrimarytRNAtranscriptsundergoaseriesofposttranscriptionalprocessingTheextrasequencesatthe5`and3`endsareremovedbyRNasePandRNaseDrespectively;TypeIVintr