分子生物學(xué)：Section K 原核生物的轉(zhuǎn)錄

回顧(review)基因表達(dá)(geneexpression)：

基因轉(zhuǎn)錄及翻譯的過(guò)程。

rRNA、tRNA的合成屬于基因表達(dá)第十一章原核生物的轉(zhuǎn)錄SectionK

TranscriptioninpokaryotesSectionK原核生物的轉(zhuǎn)錄TranscriptioninprokaryotesK1轉(zhuǎn)錄的基本原則K2大腸桿菌RNA聚合酶K3大腸桿菌σ70啟動(dòng)子K4轉(zhuǎn)錄的起始、延伸與終止

轉(zhuǎn)錄

TranscriptionThesynthesisofasingle-strandedRNAfromadouble-strandedDNAtemplate(模板).RNAsynthesisoccursinthe5’

3’directionanditssequencecorresponds(相符)tothatoftheDNAstrandwhichisknownasthesensestrand

(有義鏈).ThetemplateofRNAsynthesisistheantisensestrand(反義鏈).

Necessarycomponents:

promoter(啟動(dòng)子)/template,

RNApolymerase,rNTPs,terminator(終止子)/template(-)strandisantisensestrand反義鏈(+)strandissensestrand有義鏈K1轉(zhuǎn)錄的基本原則

起始:polymeraseandpromoter

延伸:RNApolymerase(RNA聚合酶)

終止:terminator(終止子)ATACGTATGC+1Promoter啟動(dòng)子Terminator終止子Transcribedregion轉(zhuǎn)錄區(qū)RNADNATranscriptionAntisensestrand反義鏈AUACGStructureofatranscriptionunit一個(gè)轉(zhuǎn)錄單元的結(jié)構(gòu)Whatisapromoter

(啟動(dòng)子)?ThesequenceofDNAneededforRNApolymerasetobindtothetemplateandaccomplishtheinitiationreaction

RNA聚合酶結(jié)合到模板和完成轉(zhuǎn)錄起始所需要的一段DNA序列?；蚱鹗继幍腄NA序列。起始(Initiation)Binding(捆綁)ofanRNApolymerasetothedsDNASlide(滑動(dòng))tofindthepromoterUnwind(展開(kāi))theDNAhelix(螺旋)SynthesisoftheRNAstrandatthestartsite(initiationsite),thispositioncalledposition+1轉(zhuǎn)錄泡Transcriptionbubble延伸(Elongation)Addribonucleotidestothe3’-end.TheRNApolymeraseextendthegrowingRNAchaininthedirectionof5’

3’（E.coli:40nt/sec).Theenzymeitselfmovesin3’to5’alongtheantisenseDNAstrand.延伸(Elongation)終止(Termination)Thedissociation（分離）

ofthetranscriptioncomplex(轉(zhuǎn)錄復(fù)合體)

fromthetemplatestrandandseparationofRNAstrandfromDNAOccurringattheterminator(終止子)(oftenstem-loop莖環(huán)

orhairpin發(fā)夾

structure),someneedrhoproteinasaccessoryfactor輔助因子.RNAhairpinstructure(發(fā)卡結(jié)構(gòu))5‘NNNNAAGCGCCGNNNNCCGGCGCUUUUUU-OHNNNNCG?CC?GC?GG?CC?GG?CA?UA?U…NNNNUUUU-OH2.RequiresDNAforactivityandismostactivewithadouble-strandedDNAastemplate.5’3’synthesis(NMP)n+NTP(NMP)n+1+PPiRNA聚合酶:以DNA為模板合成RNAsynthesisofRNAstrandfromDNAtemplate1.Requiresnoprimerforpolymerization3.RequireMg2+forRNAsynthesisactivity4.AllRNApolymeraseslack3’5’exonuclease核酸外切酶activity,andoneerrorusuallyoccurswhen104to105nucleotidesareincorporated合成.

噬菌體T3和T7所編碼的RNA聚合酶只有一條多肽鏈,比細(xì)菌的多亞基酶小得多。能快速合成RNA(200nt/sec)，并識(shí)別自身特異的、不同于大腸桿菌的啟動(dòng)子。5.usuallyaremultisubunitenzyme,butnotalways.7.E.colihasasingleDNA-directedRNApolymerasethatsynthesizes

alltypesofRNA.

6.DifferentfromorganismtoorganismHoloenzyme全酶:

2’forinitiation起始Coreenzyme核心酶:

2’forelongation延伸K2大腸桿菌RNA聚合酶K2大腸桿菌RNA聚合酶Bothinitiation&elongationInitiationonly36.5KD36.5KD151KD155KD11KD70KD465kd

RNAsynthesisrate:RNA合成速率

40ntpersecondat37oC

該酶是非球狀結(jié)構(gòu)。其內(nèi)有圓柱形孔道，可以直接結(jié)合16bp的DNA。整個(gè)酶可以覆蓋60bp的DNA。亞基分子量功能α36,512核心酶的組裝β150,618與轉(zhuǎn)錄過(guò)程有關(guān)(催化)β’155,613結(jié)合DNA模板(開(kāi)鏈)ω11,000未知σ70,263辨認(rèn)起始點(diǎn)大腸桿菌RNA聚合酶組分E.colipolymerase:asubunit

在核心酶中有兩個(gè)同樣的a亞基

rpoA基因編碼核心酶的組裝所必需可能在啟動(dòng)子識(shí)別中起一定作用

rpoB

基因編碼.RNA聚合酶的催化中心Rifampicin(利福平)：能與β亞基結(jié)合，RNA聚合酶的強(qiáng)抑制劑，能阻止轉(zhuǎn)錄起始但不影響延伸。不抑制真核細(xì)胞的RNA聚合酶。Streptolydigins(利迪鏈菌素)：能阻止轉(zhuǎn)錄延伸但不影響起始。3. b

亞基可能含有兩個(gè)結(jié)構(gòu)域，分別負(fù)責(zé)轉(zhuǎn)錄的起始和延伸。E.colipolymerase:bsubunit

rpoC

基因編碼.結(jié)合兩個(gè)Zn2+

離子(這兩個(gè)Zn2+

離子被認(rèn)為參與了該酶的催化作用)Heparin(肝素)：能結(jié)合到b’亞基上。在體外能抑制轉(zhuǎn)錄，并與DNA競(jìng)爭(zhēng)結(jié)合到聚合酶上.3. b’亞基可能負(fù)責(zé)酶與模板DNA的結(jié)合。E.colipolymerase:b’subunit

許多原核生物有多種s

因子，去識(shí)別不同的啟動(dòng)子序列.大腸桿菌中最常見(jiàn)的s

因子是s70.(因分子量為70KDa而得名)與s

因子的結(jié)合，使RNA聚合酶由核心酶變?yōu)槿?E.colipolymerase:sfactor

1.s

因子在啟動(dòng)子識(shí)別中起關(guān)鍵作用.

通過(guò)降低核心酶對(duì)非特異DNA序列的親和力

104倍和增加其對(duì)相應(yīng)的啟動(dòng)子序列的親和力.2.s因子對(duì)轉(zhuǎn)錄延伸并不需要.RNA鏈延伸至8-9nt時(shí)，s因子從RNA聚合酶中釋放出來(lái).s因子數(shù)量比RNA聚合酶的其它亞基的數(shù)量少.

細(xì)胞內(nèi)s因子數(shù)量只有核心酶的30%.E.colipolymerase:sfactor

K3大腸桿菌σ70

啟動(dòng)子Promotersequences:啟動(dòng)子序列

-10sequenceand-35sequenceTranscriptionstartsite

轉(zhuǎn)錄開(kāi)始位點(diǎn)3. Promoterefficiency啟動(dòng)子效率啟動(dòng)子Promoters:

RNA聚合酶特異性結(jié)合和轉(zhuǎn)錄起始所需得保守序列.ATACGTATGC+1promoterterminatorTranscribedregionRNADNATranscriptionAntisensestrand不同的啟動(dòng)子導(dǎo)致不同的轉(zhuǎn)錄起始效率，這個(gè)反過(guò)來(lái)又調(diào)控轉(zhuǎn)錄。

AUACGPromotersequences70promoter由40－60bp的序列組成.-55－+20:都被RNA聚合酶結(jié)合.-20－+20:被RNA聚合酶強(qiáng)有力保護(hù)，不受DnaseⅠ消化.

直到–40:對(duì)于啟動(dòng)子的功能仍是必需的.-10和–35序列:對(duì)于啟動(dòng)子的功能尤其重要.---5-8bp---GCTATTGACATATAAT-----16-18bp-------+1-35序列-10序列-10序列(Pribnowbox)6bpsequencewhichiscentered居中ataroundthe–10position(Pribnow,1975).AconsensussequenceofTATAATThefirsttwobases(TA)andthefinalTaremosthighlyconservedamongotherE.colipromotersThishexamer六聚體

isseparatedby5to8bpfromposition+1,andthedistanceiscriticalDNAunwinding松開(kāi)

isinitiatedatpromoterbythepolymerase-35序列:

enhances增加recognitionandinteractionwiththepolymerasesfactor

Aconservedhexamer六聚體

sequencearoundposition–35AconsensussequenceofTTGACAThefirstthreepositions(TTG)arethemostconservedamongE.colipromoters.Separatedby16-18bpfromthe–10boxin90%ofallpromotersThesequencesoffiveE.colipromotersConsensusTTGACATATAATK4轉(zhuǎn)錄的起始、

延伸、終止

Promoterbinding啟動(dòng)子結(jié)合DNAunwindingDNA解鏈

RNAchaininitiation起始RNAchainelongation延伸RNAchaintermination終止Rho-dependenttermination依靠ρ蛋白的終止Binding結(jié)合Unwinding解鏈Initiation起始Elongation延伸Termination終止啟動(dòng)子結(jié)合PromoterbindingThecoreenzyme(

2’)

hasnonspecific非特異性

DNAbinding(loosebinding,20000foldless).The

factorenhancesthespecificity特異性ofthecoreRNApolymerase(

2’)

forpromoterbinding(100x)Thepolymerasefindsthepromoter–35and–10sequencesbysliding滑行

alongtheDNAextremelyrapidlyandformingaclosedcomplex(閉合復(fù)合物)withthepromoterDNA

(Theinitialcomplexofthepolymerasewiththebase-pairedpromoterDNA)DNA解旋DNAunwindingDNA雙螺旋必須被聚合酶解旋，反義鏈才能實(shí)現(xiàn)堿基配對(duì).負(fù)超螺旋能促進(jìn)許多基因的轉(zhuǎn)錄.DNA的初始解旋導(dǎo)致DNA與聚合酶形成開(kāi)放復(fù)合物(opencomplex)，這一過(guò)程被稱(chēng)為緊密結(jié)合(

tightbinding).

RNA鏈起始

RNAchaininitiation引物不需要起始位點(diǎn)結(jié)合GTP或ATP，前者更常見(jiàn).聚合酶最先摻入最初2個(gè)核苷酸，形成磷酸二酯健.最初的9個(gè)核苷酸依次加上，酶并不沿著DNA鏈移動(dòng)，σ因子也不釋放.RNA聚合酶可能要經(jīng)過(guò)多次無(wú)效的起始(abortive

initiations)才能成功.啟動(dòng)子清除的最短時(shí)間是1-2s(一個(gè)相對(duì)較長(zhǎng)的過(guò)程)RNA鏈延伸

RNAchainelongationσ因子被釋放，形成聚合酶-DNA-新生RNA三聚復(fù)合物(ternarycomplex),使聚合酶沿DNA鏈移動(dòng)(啟動(dòng)子清除promoterclearance).轉(zhuǎn)錄泡(DNA的解旋區(qū),約17bp)和聚合酶一起沿DNA鏈移動(dòng).5’partofRNAformshybridhelix雜合雙鏈(ca.12bp)withantisenseDNAstrand.大腸桿菌聚合酶以每秒40個(gè)核苷酸速度移動(dòng).antisenseRNA鏈終止

RNAchaintermination終止Termination:dissociation分離ofRNA>re-annealing重退火

ofDNA>releaseofRNApolTerminatorsequence終止序列(stopsignal停止信號(hào)):RNAhairpinverycommonAccessory輔助rho(ρ)protein終止子Terminator一段導(dǎo)致轉(zhuǎn)錄復(fù)合體分離的特殊DNA序列.Class1:Rhoprotein(r)independent.(1)self-complementaryregionwithastem-looporhairpinsecon