RNA的生物合成课件.pptx

1、目录目录第第 十十 一一 章章Chapter 11RNA的生物合成的生物合成RNA Biosynthesis目录目录The Central DogmatranscriptiontranslationReverse transcriptionreplicationreplicationGene expression目录目录nIn the biosphere,there are two ways of synthesis of RNA:One is transcription（转录）（转录）,DNA-dependent RNA synthesis，catalyzed by DNA-dependen

2、t RNA polymeraseAnother is RNA replication,RNA-dependent RNA synthesis,catalyzed by RNA-dependent RNA polymerase，Commonly found in RNA viruses other than retroviruses(逆转录病毒）逆转录病毒）目录目录For example：Poliovirus（脊髓灰质炎病毒，（脊髓灰质炎病毒，（+）RNA）express VPg protein which covalently bind two U as primer of virus RNA

3、 replication.(+)RNA（-）RNA new（+）RNA RNA replication:RNA-dependant RNA synthesis(+)RNA(+)RNA55-pUUNNNNN-pUUNNNNNNNNAAAAAAAn 3NNNAAAAAAAn 3UUp-UUp-synthesis of（-）RNA(-)RNA(-)RNA3AANNNNNNN3AANNNNNNNNNNUUUUUUUp-NNNUUUUUUUp-5 5 -pUU-pUUsynthesis of（+）RNA目录目录转录转录 (transcription)：The synthesis of RNA molec

4、ules using DNA strands as a templates（模（模板）板）is referred to as transcription.TranscriptionRNADNA 目录目录Materials involved in transcription：substrates:NTP(ATP,UTP,GTP,CTP)template:DNA(but only one strand）enzyme:RNA polymerase,RNA-polOther protein：Rho factor(),transcriptional factors目录目录Both processes u

5、se DNA as the template.Phosphodiester bonds are formed in both cases.Both synthesis directions are from 5 to 3.Obey base pairing rules目录目录replicationtranscriptiontemplatedouble strandssingle strandsubstratedNTPNTPprimer yesno EnzymeDNA polymeraseRNA polymeraseproductdsDNAssRNAbase pairA-T,G-CA-U,T-A

6、,G-C目录目录The processes of gene expression are different in prokaryotes and eukaryotes.目录目录原核生物转录的模板和酶原核生物转录的模板和酶Templates&Enzymes in prokaryotic transcription第一节第一节Section 1 目录目录 The whole genome of DNA needs to be replicated,but only small portion of genome is transcribed in response to the developm

7、ent requirement,physiological need and environmental changes.A structural gene direct the synthesis of an RNA molecule.DNA regions that can be transcribed into RNA are called structural genes(结构基因结构基因).1.1 TemplateA B C D目录目录The template strand(模板链）模板链）is the strand from which the RNA is actually tr

8、anscribed.The coding strand（编码链）（编码链）is the strand whose base sequence specifies the amino acid sequence of the encoded protein.Only one of the two DNA strands serves as a template for RNA synthesisG C A G T A C A T G T C533C G T C A T G T A C A G5template strandcoding strandtranscriptionRNAG C A G

9、U A C A U G U C53The sequence of ribonucleotides in an RNA molecules is complementary to the sequence in one strand of DNA.N-Ala Val His Val-C peptidetranslation5335Template strandCoding strandStructural geneCoding strandTemplate strand Asymmetric transcription（不对称转录）（不对称转录）:目录目录In the two strands o

10、f a structural gene,just one strand is used as the template of transcription;（在在DNA分子双链上，一股链用作分子双链上，一股链用作模板指引转录，另一股链不转录）模板指引转录，另一股链不转录）In the case of a double-stranded DNA molecules many genes,the template strand for each gene will not necessarily be the same strand of the DNA double helix.（模板模板链并非总

11、是在同一单链上）链并非总是在同一单链上）What does Asymmetric transcription（不（不对称转录）对称转录）mean?目录目录1.2 RNA Polymerase（RNA聚合酶）聚合酶）1.2.1 RNA synthesis is catalyzed by DNA-dependent RNA polymerase.(NMP)n+NTP (NMP)n+1+ppiThe RNA product,which is synthesized in the 5 to 3 direction.目录目录目录目录 In 1955,Marianne Grunberg-Manago an

12、d Severo Ochoa reported the isolation of an enzyme that catalyzed the synthesis of RNA.For this work,Ochoa shared the 1959 Nobel Prize in Medicine with Arthur Kornberg(who received the Prize for his work on DNA polymerase I).目录目录core enzyme（核心酶）（核心酶）Holoenzyme（全酶）（全酶）1.2.2 bacterial RNA polymerase i

13、s a multi-subunits（亚基）（亚基）enzymeInitiation of transcriptionElongation of RNA chainThe subunits of E.coli RNA pol and their functionsIn prokaryotic cells,all 3 RNA classes are synthesized by a single polymerase.subunitMWfunction36512Determine the DNA to be transcribed（决定哪些基因（决定哪些基因被转录，与调控蛋白相互作用，控制转录速

14、率被转录，与调控蛋白相互作用，控制转录速率)150618Catalyze polymerization(催化作用催化作用,Rifampicin 利福平利福平(-)155613Bind&open DNA template(结合模板结合模板)70263Recognize the promoterfor synthesis initiation（识别启动子）（识别启动子）sigma factorfunction70principal sigma factor54nitrogen-regulated gene transcription32heat-shock gene transcriptionSg

15、ene expression in stationary phase cellsFexpression of flagellar operonsEinvolved in heat shock and oxidative stress responses;regulates expression of extracytoplasmic proteinsFecIregulates the fec genes for iron dicitrate transport目录目录 1.3 The initiation of transcription involves the binding RNA po

16、lymerase to promoter 转录是不连续、分区段进行的。转录是不连续、分区段进行的。每一转录区段可视为一个转录单位，称为每一转录区段可视为一个转录单位，称为操纵子操纵子(operon)。Operon includes multiple structural genes and relative upstream(上游）上游）regulatory sequences.5 3 3 5 Structure geneRegulatory regionRNA-polThe sequence that the RNA polymerase binds is called promoter.目

17、录目录调控序列中的调控序列中的promoter是是 RNA polymerase结合模结合模板板DNA的部位，也是控制转录的关键部位。原核的部位，也是控制转录的关键部位。原核生物以生物以holoenzyme结合到结合到DNA的的promoter上而起上而起动转录，其中由动转录，其中由亚基辨认亚基辨认promoter，其他亚基相，其他亚基相互配合。互配合。对启动子的研究，常采用一种巧妙的方法即对启动子的研究，常采用一种巧妙的方法即RNA聚合酶保护法（聚合酶保护法（RNA pol protection）。RNA pol protection5 5 Structure geneconservativ

18、e sequence consensus sequencetranscriptionT T G A C AA A C T G T-35(Pribnow box)T A T A A T Pu A T A T T A Py-10 1-30-5 010-10-40-205 3 3 5 (Recognition site)RNA-pol辨认位点辨认位点目录目录Frequency in 45 samples 38 36 29 40 25 30 37 37 28 41 29 44T T G A C AT A T A A T目录目录目录目录nRNA polymerase holoenzyme在转录起始区的结

19、合在转录起始区的结合:目录目录原核生物的转录过程原核生物的转录过程The Process of Transcription in Prokaryote第二节第二节目录目录General concepts Three phases:initiation（起始）,elongation（延长）,and termination（终止）.The prokaryotic RNA-pol can bind to the DNA template directly in the transcription process.The eukaryotic RNA-pol requires co-factors（辅

20、助因子）to bind to the DNA template together in the transcription process.目录目录Transcription of ProkaryotesInitiation phase（起始阶段）:RNA-pol recognizes the promoter and starts the transcription.Elongation phase（延长阶段）:the RNA strand is continuously growing.Termination phase（终止阶段）:the RNA-pol stops synthesis

21、and the nascent RNA is separated from the DNA template.目录目录 RNA聚合酶必须准确地结合在转录模板的聚合酶必须准确地结合在转录模板的起始区域。起始区域。DNA双链解开，使其中的一条链作为转录双链解开，使其中的一条链作为转录的模板。的模板。2.1 Initiation of transcription needs RNA polymerase holoenzyme(全酶全酶)n转录起始需解决两个问题：转录起始需解决两个问题：目录目录 The holoenzyme binds with the DNA;RNA-pol recognizes

22、the TTGACA region(-35区),and slides to the TATAAT(-10区)Unwinds the DNA,and transcriptional bubble is formed;The first polymerization reaction catalized by RNA pol;5-pppG-OH +NTP 5-pppGpN OH+ppi The initiation complex is formed.pppGNTPpppGpN-OHppiRNA-pol(2)-DNA-pppGpN-OH 3 目录目录 No primer(引物）is needed

23、for RNA synthesis.The subunit falls off from the RNA-pol once the first 3,5 phosphodiester bond is formed.The core enzyme（核心酶）moves along the DNA template to enter the elongation phase.2.DNA双链局部解开，形成双链局部解开，形成开放转录复合体开放转录复合体(open transcription complex)；1.RNA聚合酶全酶聚合酶全酶(2)与模板结合，形成与模板结合，形成闭合转录复闭合转录复合体合体(

24、closed transcription complex)；3.在在RNA聚合酶作用下发生第一次聚合反应，形成转录聚合酶作用下发生第一次聚合反应，形成转录起始复合物：起始复合物：RNApol(2)-DNA-pppGpN-OH 3 5-pppG-OH +NTP 5-pppGpN-OH 3 +ppin转录起始过程：转录起始过程：4.亚基即从转录起始复合物上脱落，核心酶连同四磷酸二亚基即从转录起始复合物上脱落，核心酶连同四磷酸二核苷酸，继续结合于核苷酸，继续结合于DNA模板上，酶沿模板上，酶沿DNA链前移，进链前移，进入延长阶段。入延长阶段。目录目录目录目录2.2 in Prokaryote，Elo

25、ngation of transcription and translation at the same time The release of the subunit causes the conformational change（构象改变）（构象改变）of the core enzyme.The core enzyme slides on the DNA template toward the 3 end.Free NTPs are added sequentially to the 3 -OH of the nascent RNA strand.Transcription bubble

26、目录目录 RNA-pol,DNA segment of 40nt and the nascent RNA form a complex called the transcription bubble（转录空泡）（转录空泡）.The 3 segment of the nascent RNA hybridizes with the DNA template,and its 5 end extends out the transcription bubble as the synthesis is processing.转录空泡转录空泡(transcription bubble)：RNA-pol（核

27、心酶）（核心酶）DNA RNA目录目录RNA-pol of E.ColiRNA-pol of E.Coli目录目录Simultaneous transcriptions and translation5 3 DNAn原核生物转录过程中的羽毛状现象原核生物转录过程中的羽毛状现象核糖体核糖体RNARNA聚合酶聚合酶在同一在同一DNA模板上，有多个转录同时在进行；模板上，有多个转录同时在进行；转录尚未完成，翻译已在进行。转录尚未完成，翻译已在进行。这种形状说明：这种形状说明：目录目录 Rho dependent termination,依赖依赖Rho 因子的转录终止因子的转录终止 Rho indep

28、endent termination,非依赖非依赖Rho因子的转录终止因子的转录终止2.3 Termination of transcription has two ways:Rho-dependent and Rho-independent way.RNA pol stops and doesnt move again and nascent RNA strand is released from the transcription complex,then the core polymerase dissociates.(1)The termination function of fact

29、orThe factor,a hexamer（46KD）,is a ATPase and a helicase，can bind to RNA，especially to poly C.目录目录n因子的作用原理：因子的作用原理：factor and RNA pol change their conformation，RNA pol pause and helicase and ATPase activity of Rho induce dissociation of RNA pol and DNA/RNA hybrid segment.目录目录目录目录 The termination sign

30、al（终止信号）（终止信号）is a stretch of 30-40 nucleotides on the RNA transcript,consisting of many GC followed by a series of U.The sequence specificity of this nascent RNA transcript will form particular stem-loop structures(茎茎-环结构）环结构）to terminate the transcription.5UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUA

31、GUCACCAGCCUUUUU.3 5UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU.3 RNA 5 TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT.3 DNA UUUU.UUUU.5UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU.3近终止区的转录产物形成发夹近终止区的转录产物形成发夹(hairpin)结构结构是非依赖是非依赖因子终止的普遍现象。因子终止的普遍现象。目录目录 The stem-loo

32、p structure alters the conformation of RNA-pol,leading to the pause of the RNA-pol moving.Then the competition of the RNA-RNA hybrid and the DNA-DNA hybrid reduces the DNA-RNA hybrid stability,and causes the transcription complex dissociated.Among all the base pairings,the most unstable one is rU:dA

33、.茎环结构使转录终茎环结构使转录终止的机理）止的机理）目录目录目录目录目录目录目录目录目录目录termination目录目录真核生物的转录过程真核生物的转录过程The Process of Transcription in Eukaryote第三节第三节目录目录Roger D.KornbergArthur Kornberg（1959）（1918-2007）(right 2)Ken B.KornbergThomas B.Kornberg目录目录真核生物的转录过程比原核复杂。二者的转录真核生物的转录过程比原核复杂。二者的转录起始过程有较大区别，转录终止也不相同。起始过程有较大区别，转录终止也不相同

34、。Three phases:initiation（起始）,elongation（延长）,and termination（终止）.The prokaryotic RNA-pol can bind to the DNA template directly in the transcription process.The eukaryotic RNA-pol requires co-factors（辅助因子）to bind to the DNA template together in the transcription process.目录目录3.1 Eukaryotic systems have

35、 three kinds of RNA polymerases.（真核生物有三种真核生物有三种DNA依依赖性赖性RNA聚合酶）聚合酶）n Three kinds of is responsible for transcription of different RNAs,but lack a proofreading function（缺乏校读功能）（缺乏校读功能）.RNA聚合酶聚合酶（RNA Pol）RNA聚合酶聚合酶（RNA Pol）RNA聚合酶聚合酶（RNA Pol）目录目录RNA-polIIIIII products45S rRNAhnRNA5S rRNAtRNA snRNASensit

36、ivity to AmanitinNohighmoderateRNA-pol of eukaryotesAmanitin(鹅膏鹅膏蕈碱蕈碱)is a specific inhibitor of RNA-pol.目录目录 Two large subunits(similar to subunit)and 12-15 smaller subunits.The largest subunit of RNA pol is RBP1which has a long carboxyl-terminal domain(羧基末端结构域羧基末端结构域,CTD)consisting of many repeats

37、 of a consensus sequence(共有序列共有序列)-Tyr-Ser-Pro-Thr-Ser-Pro-Ser(21 in yeast,52 in mouse and human).CTD is essential to cell survival.However,the eukaryotic RNA pol does not contain any subunit similar to factor.Therefore,the initiation of transcription is mediated by other proteins.n each of RNA pol

38、is a multiple-subunit protein and its structure is more complex than those in prokaryotic cell.目录目录3.2 启启动子）动子）转录因子）转录因子）（1 1）the core promoter element of Transcription initiation upstream section The cis-acting elements（顺式作用元件）顺式作用元件）are the specific sequences on the DNA template that regulate the

39、transcription of one or more genes.目录目录 cis-acting elements include promoter,upstream promoter elements(or promoter-proximal elements,启动子上游元件启动子上游元件)and enhancer（增强子（增强子,a distant element）.起始点上游多数有共同的起始点上游多数有共同的TATA序列，称为序列，称为Hognees盒盒或或TATA盒盒(TATA box)。位于转录起始点附近的起始。位于转录起始点附近的起始子子(intiator，Inr)。通常认为。

40、通常认为TATA box and initiator is core promoter element（启动子的核心序列）（启动子的核心序列）。53调控序列调控序列TATA盒盒InrYYAN YYTA-30+1TATAAATATA box Similar to -10 region（70recognition）structural geneGCGCCAATTATAintronexonexonstartCAAT boxGCboxenhancercis-acting elementTATA box(Hogness box)Cis-acting element目录目录nRNA pol:hnRNA目录

41、目录RNA Pol I Transcript products:45s rRNA Promoter elements:核心启动子核心启动子(core promoter)和和上游控制元件上游控制元件(upstream control element,UCE)。目录目录RNA Pol III Transcript products：small molecule RNA(tRNA,5S rRNA and partial snRNA)Promoter is located downstream of transcription start，named as internal control regio

42、ns(ICR,内部控制内部控制区区).目录目录 RNA-pol does not bind the promoter directly.RNA-pol II associates with six transcription factors,TFII A-H.The trans-acting factors（反式作用因子）（反式作用因子）are the proteins（蛋白质蛋白质）that recognize and bind（识别并识别并结合结合）directly or indirectly cis-acting elements（顺顺式作用元件式作用元件）and regulate it

43、s activity.Trans-acting factors which can bind to RNA pol directly or in directly are called transcriptional factors（转录因子，（转录因子，TF）.（2）Transcription factors，转录因子转录因子参与参与RNA-pol转录的转录的TF蛋白激酶活性，使蛋白激酶活性，使CTD磷磷酸化酸化TFHATPase57（）34（）TFE解螺旋酶解螺旋酶30，74TFF促进促进RNA-pol结合及作结合及作为其他因子结合的桥梁为其他因子结合的桥梁33TFB稳定稳定TFD-DNA

44、复合物复合物12，19，35TFA辅助辅助TBP-DNA结合结合TAF*结合结合TATA盒盒TBP*38TFD功功能能亚基组成，分子量亚基组成，分子量(kD)转录因子转录因子蛋白激酶活性，使蛋白激酶活性，使CTD磷磷酸化酸化TFHATPase57（）34（）TFE解螺旋酶解螺旋酶30，74TFF促进促进RNA-pol结合及作结合及作为其他因子结合的桥梁为其他因子结合的桥梁33TFB稳定稳定TFD-DNA复合物复合物12，19，35TFA辅助辅助TBP-DNA结合结合TAF*结合结合TATA盒盒TBP*38TFD功功能能亚基组成，分子量亚基组成，分子量(kD)转录因子转录因子目录目录型基因中的四

45、类转录因子型基因中的四类转录因子 转录因子转录因子 具体组分具体组分 结合序列结合序列 功能功能 基本组分基本组分 TBP,TFA,B,E,G,F和和H TBP结合结合TATA盒盒 转录起始定位；转录起始定位；转转录起始和延长录起始和延长 辅激活因子辅激活因子 TAFs和中介和中介子子 在可诱导因子和上游在可诱导因子和上游因子与基本转录因子、因子与基本转录因子、RNA聚合酶结合中聚合酶结合中起联结和中介作用起联结和中介作用 上游因子上游因子 SP1、ATF、CTF等等 启动子上游启动子上游元件元件 协助基本转录因子，协助基本转录因子，提高转录效率和专一提高转录效率和专一性性 可诱导因子可诱导因

46、子 如如MyoD、HIF-1等等 增强子等远增强子等远隔调控序列隔调控序列 时间和空间（组织）时间和空间（组织）特异性地调控转录特异性地调控转录 目录目录RNA聚合酶聚合酶II与启动子的结合、启动转录需要多种与启动子的结合、启动转录需要多种蛋白质因子的协同作用，包括：蛋白质因子的协同作用，包括：（1）可诱导因子或上游因子与增强子或启动子上游）可诱导因子或上游因子与增强子或启动子上游元件的结合；元件的结合；（2）通用转录因子在启动子处的组装；辅激活因子）通用转录因子在启动子处的组装；辅激活因子和和/或中介子在通用转录因子或中介子在通用转录因子/RNA聚合酶聚合酶II复合物与复合物与可诱导因子、上

47、游因子之间的辅助和中介作用。可诱导因子、上游因子之间的辅助和中介作用。（3）因子和因子之间互相辨认、结合，以准确地控）因子和因子之间互相辨认、结合，以准确地控制基因是否转录、何时转录。制基因是否转录、何时转录。目录目录Promoter and regulatory proteinsTF for eukaryotic transcription目录目录（3）Pre-initiation complex(PIC)，转录起始前复合物转录起始前复合物真核生物真核生物RNA-pol不与不与DNA分子直接结合，而需分子直接结合，而需依靠众多的转录因子，形成依靠众多的转录因子，形成转录起始复合物转录起始复合

48、物(pre-initiation complex,PIC)。TBP of TFII D binds TATA TFII A and TFII B bind TFII D TFII F-RNA-pol complex binds TFII B TFII F and TFII E open the dsDNA(helicase and ATPase)TFII H:completion of PIC Pre-initiation complex(PIC)RNA pol IITF II FTBP TAFTATADNATF II ATF II BTF II ETF II H目录目录目录目录 TF II

49、H is of protein kinase activity to phosphorylate CTD of RNA-pol.(CTD is the C-terminal domain of RNA-pol)Only the p-RNA-pol can move toward the downstream,starting the elongation phase.Most of the TFs fall off from PIC during the elongation phase.*Phosphorylation（磷酸化）（磷酸化）of RNA-pol目录目录nPIC的形成的形成目录目

50、录（4）A small number of trans-acting factors in the transcription of specific genes with start 为了保证转录的准确性，不同基因需不同转录因子。为了保证转录的准确性，不同基因需不同转录因子。拼板理论拼板理论(piecing theory)：several trans-acting factors（mainly inducible factors and upstream factors,可诱导可诱导因子和上游因子因子和上游因子）bind together to form active and specifi