RNA的生物合成课件

1、第 十 一 章Chapter 11RNA的生物合成RNA Biosynthesis第 十 一 章RNA的生物合成The Central DogmatranscriptiontranslationReverse transcriptionDNARNAproteinreplicationreplicationGene expressionThe Central DogmatranscriptiIn the biosphere, there are two ways of synthesis of RNA :One is transcription（转录）, DNA-dependent RNA sy

2、nthesis， catalyzed by DNA-dependent 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） In the biosphere, there are twFor example：Poliovirus（脊髓灰质炎病毒，（+）RNA）express VPg protein w

3、hich covalently bind two U as primer of virus RNA replication. (+)RNA （-）RNA new（+）RNA RNA replication: RNA-dependant RNA synthesis(+) RNA5-pUUNNNNNNNNAAAAAAAn 3UUp-synthesis of（-）RNA(-) RNA3AANNNNNNNNNNUUUUUUUp- 5 -pUUsynthesis of （+）RNAFor example：Poliovirus（脊髓灰质炎病毒转录 (transcription) ： The synthes

4、is of RNA molecules using DNA strands as a templates（模板） is referred to as transcription.The genetic information can be transferred from DNA to RNA.TranscriptionRNADNA 转录 (transcription) ： The synthMaterials involved in transcription：substrates: NTP (ATP, UTP, GTP, CTP) template: DNA ( but only one

5、strand）enzyme: RNA polymerase, RNA-polOther protein：Rho factor(), transcriptional factorsMaterials involved in transcrBoth processes use DNA as the template.Phosphodiester bonds are formed in both cases. Both synthesis directions are from 5 to 3.Obey base pairing rulesSimilarity between replication

6、and transcriptionBoth processes use DNA as the replicationtranscriptiontemplatedouble strandssingle strandsubstratedNTPNTPprimer yesno EnzymeDNA polymeraseRNA polymeraseproductdsDNAssRNAbase pairA-T, G-CA-U, T-A, G-CDifferences between replication and transcription replicationtranscriptiontemplThe p

7、rocesses of gene expression are different in prokaryotes and eukaryotes.The processes of gene expressi原核生物转录的模板和酶Templates & Enzymes in prokaryotic transcription第一节Section 1 原核生物转录的模板和酶 The whole genome of DNA needs to be replicated, but only small portion of genome is transcribed in response to the

8、 development 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).1.1 TemplateA B C D The whole genome of DNA needsThe template strand (模板链）is the strand f

9、rom which the RNA is actually transcribed. The coding strand（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 synthesisThe template strand (模板链）is thThe sequence of ribonucleotides in an RNA

10、molecules is complementary to the sequence in one strand of DNA.N- Ala Val His Val-C peptidetranslationThe sequence of ribonucleotide5335Template strandCoding strandStructural geneCoding strandTemplate strand Asymmetric transcription （不对称转录）:53Template strandCoding str In the two strands of a struct

11、ural gene, just one strand is used as the template of transcription;（在DNA分子双链上，一股链用作模板指引转录，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. （ 模板链并非总是在同一单链上）What does Asymmetric trans

12、cription（不对称转录） mean? In the two strands of a struc1.2 RNA Polymerase（RNA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.1.2 RNA Polymerase（RNA聚合酶） 1The prokaryotic（原核） RNA polymerase is a

13、multiple-subunit protein of 480kD. RNA polymerase does not require a primer (引物） to initiate synthesis.RNA polymerase initiate RNA synthesis by binding a special DNA sequence promoter（启动子）.The prokaryotic（原核） RNA polymeDiscovery of RNA polymeraseIn 1955, Marianne Grunberg-Manago and Severo Ochoa rep

14、orted 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). Discovery of RNA polymeraseIn core enzyme（核心酶）Holoenzyme（全酶） 1.2.2 bacterial RNA polymera

15、se is a multi-subunits （亚基）enzymeInitiation of transcriptionElongation of RNA chaincore enzyme（核心酶）Holoenzyme（全酶）The 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

16、（决定哪些基因被转录，与调控蛋白相互作用，控制转录速率)150618Catalyze polymerization (催化作用, Rifampicin 利福平(-)155613Bind & open DNA template(结合模板)70263Recognize the promoterfor synthesis initiation（识别启动子）The subunits of E.coli RNA poSeven alternative sigma factors of E. coli.sigma factorfunction70principal sigma factor54nitrog

17、en-regulated gene transcription32heat-shock gene transcriptionSgene 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 transportSev

18、en alternative sigma factor 1.3 The initiation of transcription involves the binding RNA polymerase to promoter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，称为操纵子(operon)。Operon includes multiple structural genes and relative upstream(上游） regulatory sequences. 5335Structure geneRegulatory regionRNA-polThe sequence

19、that the RNA polymerase binds is called promoter. 1.3 The initiation of transc调控序列中的promoter是 RNA polymerase结合模板DNA的部位，也是控制转录的关键部位。原核生物以holoenzyme结合到DNA的promoter上而起动转录，其中由亚基辨认promoter，其他亚基相互配合。 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（ RNA pol protection ）。调控序列中的promoter是 RNA polymeraseRNA pol protection55Struc

20、ture geneRNA pol protection55Structurconservative 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conservative sequence transcriConsensus SequenceFrequency in 45 samples 38 36 29

21、 40 25 30 37 37 28 41 29 44T T G A C AT A T A A TConsensus SequenceFrequency inRNA的生物合成课件RNA polymerase holoenzyme在转录起始区的结合:RNA polymerase holoenzyme在转录起始原核生物的转录过程The Process of Transcription in Prokaryote第二节原核生物的转录过程第二节 General conceptsThree phases: initiation（起始）, elongation（延长）, and termination（终

22、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. General conceptsThree phases: Transcription of ProkaryotesInitiation phase（起始阶段）: RNA-po

23、l 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 and the nascent RNA is separated from the DNA template. Transcription of ProkaryotesIRNA聚合酶必须准确地结合在转录模板的起始区域。DNA双链解开，使其中的一条链作为转

24、录的模板。2.1 Initiation of transcription needs RNA polymerase holoenzyme (全酶)转录起始需解决两个问题：RNA聚合酶必须准确地结合在转录模板的起始区域。2.1 InThe holoenzyme binds with the DNA; RNA-pol recognizes the TTGACA region(-35区), and slides to the TATAAT(-10区) Unwinds the DNA, and transcriptional bubble is formed;The first polymerizat

25、ion reaction catalized by RNA pol; 5-pppG -OH + NTP 5 -pppGpN OH + ppiThe initiation complex is formed.pppGNTPpppGpN - OHppiRNA-pol (2) - DNA - pppGpN- OH 3 The holoenzyme binds with the No primer (引物）is needed for RNA synthesis. The subunit falls off from the RNA-pol once the first 3,5 phosphodiest

26、er bond is formed. The core enzyme（核心酶） moves along the DNA template to enter the elongation phase. No primer (引物）is needed for RN2. DNA双链局部解开，形成开放转录复合体(open transcription complex) ；1. RNA聚合酶全酶(2)与模板结合，形成闭合转录复合体(closed transcription complex) ；3. 在RNA聚合酶作用下发生第一次聚合反应，形成转录起始复合物：RNApol (2) - DNA - pppGp

27、N- OH 35-pppG -OH + NTP 5-pppGpN - OH 3 + ppi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核苷酸，继续结合于DNA模板上，酶沿DNA链前移，进入延长阶段。 2. DNA双链局部解开，形成开放转录复合体(open trRNA的生物合成课件 2.2 in Prokaryote ，Elongation of transcription and translation at the same time The release of the subunit causes the conformational change （构象改变）o

28、f 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. 2.2 in Prokaryote ，Elongatio Transcription bubble Transcription bubble RNA-pol, DNA segment of 40nt and the nascent RNA form a complex called the t

29、ranscription bubble（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(transcription bubble)：RNA-pol （核心酶） DNA RNA RNA-pol, DNA segment of 40nt RNA-pol of E. Coli RNA-pol of E. Coli RNA-pol

30、 of E. Coli RNA-pol of E. ColiSimultaneous transcriptions and translationSimultaneous transcriptions an53DNA原核生物转录过程中的羽毛状现象核糖体RNARNA聚合酶在同一DNA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。 这种形状说明：53DNA原核生物转录过程中的羽毛状现象核糖体RNARNRho dependent termination,依赖Rho 因子的转录终止Rho independent termination,非依赖Rho因子的转录终止2.3 Terminatio

31、n of transcription has two ways: Rho-dependent and Rho-independent way.Termination means that RNA pol stops and doesnt move again and nascent RNA strand is released from the transcription complex, then the core polymerase dissociates. RNA聚合酶在DNA模板上停顿下来不再前进，转录产物RNA链从转录复合物上脱落下来。 Rho dependent terminat

32、ion,依赖Rh (1) The termination function of factorThe factor, a hexamer（46KD）, is a ATPase and a helicase，can bind to RNA ，especially to poly C. (1) The termination function 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

33、 RNA pol and DNA/RNA hybrid segment.因子的作用原理：factor and RNA pol cRNA的生物合成课件(2) -independent termination，非依赖Rho因子的转录终止The termination signal（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 tran

34、script will form particular stem-loop structures (茎-环结构）to terminate the transcription.(2) -independent termination，5UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU. 3 5UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU. 3 RNA 5TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT.

35、 3 DNA UUUU. UUUU.5UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU. 3近终止区的转录产物形成发夹(hairpin)结构是非依赖因子终止的普遍现象。 rplL protein5UUGCAGCCUGACAAAUCAGGCUGAUGGC The stem-loop structure alters the conformation of RNA-pol, leading to the pause of the RNA-pol moving.Then the competition of the RNA-RNA hy

36、brid 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. Stem-loop disruption（茎环结构使转录终止的机理） The stem-loop structure alter RNA的生物合成课件 terminationtermination真核生物的转录过程The Process of T

37、ranscription in Eukaryote第三节真核生物的转录过程第三节Science familyRoger D. Kornberg （2006）(left 1)Arthur Kornberg（1959）（1918-2007）(right 2)Ken B. Kornberg (left 2)Thomas B. Kornberg (right 1)Science familyRoger D. Kornber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。Three phases: initiation（起始）, elongation（延长）, and te

38、rmination（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. 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，3.1 Eukaryotic systems have three kinds of RNA

39、polymerases. （真核生物有三种DNA依赖性RNA聚合酶）Three kinds of is responsible for transcription of different RNAs, but lack a proofreading function （缺乏校读功能）.RNA聚合酶（RNA Pol）RNA聚合酶（RNA Pol）RNA聚合酶（RNA Pol ）3.1 Eukaryotic systems have tRNA-polIIIIII products45S rRNAhnRNA5S rRNAtRNA snRNASensitivity to AmanitinNohighm

40、oderateRNA-pol of eukaryotesAmanitin(鹅膏蕈碱)is a specific inhibitor of RNA-pol. RNA-polIIIIII products45S rRNTwo 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, CTD ) consisting of many repeats of

41、a consensus sequence (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. each of RNA pol i

42、s a multiple-subunit protein and its structure is more complex than those in prokaryotic cell.Two large subunits (similar to3.2 Transcription initiation needs promoter（启动子） ，upstream regulatory regions（上游调控元件） ，RNA pol and transcription factor （转录因子）.（1）the core promoter element of Transcription ini

43、tiation upstream sectionThe cis-acting elements（顺式作用元件）are the specific sequences on the DNA template that regulate the transcription of one or more genes. 3.2 Transcription initiation cis-acting elements include promoter, upstream promoter elements (or promoter-proximal elements, 启动子上游元件) and enhan

44、cer（增强子, a distant element）.起始点上游多数有共同的TATA序列，称为Hognees盒或TATA盒(TATA box)。位于转录起始点附近的起始子(intiator，Inr)。通常认为TATA box and initiator is core promoter element（启动子的核心序列）。 53调控序列TATA盒InrYYAN YYTA-30+1TATAAAcis-acting elements include pr TATA box Similar to -10 region（70recognition） TATA box Similar to -10 r

45、egi Cis-acting element Cis-acting elementRNA pol : hnRNARNA pol : hnRNARNA Pol I Transcript products: 45s rRNAPromoter elements: 核心启动子(core promoter)和上游控制元件(upstream control element, UCE)。 RNA Pol I Transcript products:RNA Pol IIITranscript products：small molecule RNA (tRNA, 5S rRNA and partial snRN

46、A)Promoter is located downstream of transcription start ，named as internal control regions ( ICR,内部控制区). RNA Pol IIITranscript productsRNA-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）are the proteins（蛋白质）

47、 that recognize and bind （识别并结合）directly or indirectly cis-acting elements （顺式作用元件）and regulate its activity. Trans-acting factors which can bind to RNA pol directly or in directly are called transcriptional factors（转录因子， TF）.（2）Transcription factors，转录因子RNA-pol does not bind the prom参与RNA-pol转录的TF参

48、与RNA-pol转录的TF型基因中的四类转录因子 转录因子 具体组分 结合序列 功能 基本组分 TBP,TFA,B,E,G,F和H TBP结合TATA盒 转录起始定位； 转录起始和延长 辅激活因子 TAFs和中介子 在可诱导因子和上游因子与基本转录因子、RNA聚合酶结合中起联结和中介作用 上游因子 SP1、ATF、CTF等 启动子上游元件 协助基本转录因子，提高转录效率和专一性 可诱导因子 如MyoD、HIF-1等 增强子等远隔调控序列 时间和空间（组织）特异性地调控转录 型基因中的四类转录因子 转录因子 具体组分 结合序列 功能RNA聚合酶II与启动子的结合、启动转录需要多种蛋白质因子的协同

49、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；辅激活因子和/或中介子在通用转录因子/RNA聚合酶II复合物与可诱导因子、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，以准确地控制基因是否转录、何时转录。RNA聚合酶II与启动子的结合、启动转录需要多种蛋白质因子的 Promoter and regulatory proteins Promoter and regulatory proteTF for eukaryotic transcriptionTF for eukaryotic transcriptio（3） P

50、re-initiation complex (PIC)，转录起始前复合物真核生物RNA-pol不与DNA分子直接结合，而需依靠众多的转录因子，形成转录起始复合物(pre-initiation complex, PIC) 。 TBP of TFII D binds TATA TFII A and TFII B bind TFII DTFII F-RNA-pol complex binds TFII BTFII F and TFII E open the dsDNA (helicase and ATPase)TFII H: completion of PIC （3） Pre-initiation

51、complex (PIPre-initiation complex (PIC)TBPTAFTF II HPre-initiation complex (PIC)TBRNA的生物合成课件 TF II 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 T

52、Fs fall off from PIC during the elongation phase. * Phosphorylation（磷酸化） of RNA-pol TF II H is of protein kinase PIC的形成PIC的形成（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。拼板理论(piecing theory) ： several trans-acting factors（mainly i

53、nducible factors and upstream factors ,可诱导因子和上游因子） bind together to form active and specific complex which can bind general TF and RNA pol directly or by co-activators or mediator(辅激活因子或中介子)and initate to transcribe a specific gene.（4）A small number of trans-actRNA的生物合成课件 Promoter and regulatory pro

54、teins Promoter and regulatory prote3.3 Eukaryotic transcription elongation process of transcription and translation are not synchronous phenomena The elongation is similar to that of prokaryotes. The transcription and translation （翻译）cant couple（偶联） since they are separated by nuclear membrane（核膜）.

55、As RNA-pol move, it may reach nucleosome（核小体）. You can observe the movement and degradation of nucleosome.3.3 Eukaryotic transcription RNA-PolRNA-PolRNA-Polnucleosome转录延长中的核小体移位Direction of transcriptionRNA-PolRNA-PolRNA-Polnucleosom3.4 Eukaryotic transcription termination and post-transcriptional m

56、odification at the same timeThe termination is closely related to the post-transcriptional modification（转录后修饰）.Poly (A) tail is added to the 3end of mRNA after post-transcription.The termination sequence is AATAAA （Modification points, 转录终止的修饰点）， followed by GT repeats.3.4 Eukaryotic transcription 真

57、核生物的转录终止及加尾修饰真核生物的转录终止及加尾修饰真核生物RNA的加工Post-transcriptional Modification of Eukaryotic RNA第四节真核生物RNA的加工第四节The nascent RNA（新生RNA）, also known as primary transcript（初级转录产物）, needs to be modified to become functional tRNAs, rRNAs, and mRNAs. The posttranscriptional processing (转录后加工）occurs in both prokar

58、yotes and eukaryotes. The most notable difference between the two organisms are mainly in the processing of mRNA.The nascent RNA（新生RNA）, also kRNA的生物合成课件Major modification modes：1. 剪接(splicing)2. 剪切(cleavage)3. 修饰(modification)4. 添加(addition)Major modification modes：1. 剪接Primary transcripts of mRNA

59、are called as heterogeneous nuclear RNA (hnRNA). hnRNA are larger than matured mRNA by many folds. Modification includes Capping at the 5- end Tailing at the 3- endmRNA splicing（剪接）RNA edition （编辑） 一、真核生物mRNA的加工包括首、尾修饰和剪接Primary transcripts of mRNA ar（一）adding a cap to the 5-end of a pre-mRNA 在5-末端加

60、入“帽”结构 5 cap structure (m7GpppGp -) Cap formation is catalyzed by capping enzyme (加帽酶) and methyltransferase(甲基转移酶) . （一）adding a cap to the 5-end Capping at the 5- endm7GpppGp- Capping at the 5- endm7GpppG The 5- cap structure is found on hnRNA too. The capping process occurs in nuclei. The cap str