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1、第 十 一 章Chapter 11,RNA的生物合成RNA Biosynthesis,第 十 一 章RNA的生物合成,The Central Dogma,transcription,translation,Reverse transcription,DNA,RNA,protein,replication,replication,Gene expression,The Central Dogmatranscripti,In the biosphere, there are two ways of synthesis of RNA :,One is transcription（转录）, DNA-d
2、ependent RNA synthesis， 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 tw,For example：Poliovirus（脊髓灰质炎病毒，（+）RNA）expre
3、ss VPg protein which covalently bind two U as primer of virus RNA replication. (+)RNA （-）RNA new（+）RNA,RNA replication: RNA-dependant RNA synthesis,For example：Poliovirus（脊髓灰质炎病毒,转录 (transcription) ： The synthesis of RNA molecules using DNA strands as a templates（模板） is referred to as transcription.
4、,The genetic information can be transferred from DNA to RNA.,Transcription,转录 (transcription) ： The synth,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,M
5、aterials involved in transcr,Both processes use DNA as the template.Phosphodiester bonds are formed in both cases. Both synthesis directions are from 5 to 3.Obey base pairing rules,Similarity between replication and transcription,Both processes use DNA as the,Differences between replication and tran
6、scription,replicationtranscriptiontempl,The processes 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 por
7、tion of genome is transcribed in response to the 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 Template,The whole genome of DNA nee
8、ds,The template strand (模板链）is the strand from 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 synthesis,The template strand (模板链）is
9、th,The sequence of ribonucleotides in an RNA molecules is complementary to the sequence in one strand of DNA.,N- Ala Val His Val-C peptide,translation,The sequence of ribonucleotide,Asymmetric transcription （不对称转录）:,53Template strandCoding str,In the two strands of a structural gene, just one strand
10、 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 transcription（不对称转录） mean?,In
11、the two strands of a struc,1.2 RNA Polymerase（RNA聚合酶）, 1.2.1 RNA synthesis is catalyzed by DNA-dependent RNA polymerase.,(NMP)n + NTP (NMP)n+1 + ppi,The RNA product, which is synthesized in the 5 to 3 direction.,1.2 RNA Polymerase（RNA聚合酶） 1,The prokaryotic（原核） RNA polymerase is a multiple-subunit pr
12、otein 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 polyme,Discovery of RNA polymerase,In 1955, Marianne Grunberg-Manago and Severo Ochoa reported the isolat
13、ion 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 polymerase is a multi-
14、subunits （亚基）enzyme,Initiation of transcription,Elongation of RNA chain,core enzyme（核心酶）Holoenzyme（全酶）,The subunits of E.coli RNA pol and their functions,In prokaryotic cells, all 3 RNA classes are synthesized by a single polymerase.,The subunits of E.coli RNA po,Seven alternative sigma factors of E
15、. coli.,Seven 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.,The sequence that the RNA polymerase binds
16、 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 polymerase,RNA pol protection,RNA pol protection55Structur,co
17、nservative sequence consensus sequence,( Recognition site ),RNA-pol辨认位点,conservative sequence transcri,Consensus Sequence,Frequency in 45 samples 38 36 29 40 25 30 37 37 28 41 29 44,T T G A C A,T A T A A T,Consensus SequenceFrequency in,RNA的生物合成课件,RNA polymerase holoenzyme在转录起始区的结合:,RNA polymerase h
18、oloenzyme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 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-facto
19、rs （辅助因子）to bind to the DNA template together in the transcription process.,General conceptsThree phases:,Transcription of Prokaryotes,Initiation 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 pha
20、se（终止阶段）: the RNA-pol stops synthesis and the nascent RNA is separated from the DNA template.,Transcription of ProkaryotesI,RNA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 In,The holoenzyme
21、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 polymerization reaction catalized by RNA pol; 5-pppG -OH + NTP 5 -pppGpN OH + ppiThe initiation complex is formed.,pppG,NTP,pppGpN - OH,RNA-p
22、ol (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 phosphodiester 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 R
23、N,2. 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 - pppGpN- OH 3,5-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
24、解开，形成开放转录复合体(open tr,RNA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）of the core enzyme. The core enzyme slides on the DNA template toward the 3 end. Free NTPs are added sequentially to the 3
25、 -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 transcription bubble（转录空泡）. The 3 segment of the nascent RNA hybridizes with the DNA template, and its 5 end extends out t
26、he 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 of E. Coli,RNA-pol of E. Coli,Simultaneous transcriptions and translation,Simultaneous transcriptions an,5,3,DNA,原核生物转录
27、过程中的羽毛状现象,核糖体,RNA,RNA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RNARN,Rho 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 Termination of transcription has two ways: Rho-dependent and Rho-independent way.,Termination means that RNA pol stop
28、s 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 termination,依赖Rh,(1) The termination function of factor,The factor, a hexamer（46KD）, is a ATPase and a helicase，ca
29、n 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 RNA pol and DNA/RNA hybrid segment.,因子的作用原理：factor and RNA pol c,RNA的生物合成课件,(2) -independent termination
30、，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 transcript will form particular stem-loop structures (茎-环结构）to terminate the transcription.,(2) -independ
31、ent termination，,5UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU. 3,5UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU. 3,RNA,5TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT. 3,DNA,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
32、止的普遍现象。,rplL protein,5UUGCAGCCUGACAAAUCAGGCUGAUGGC,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 hybrid and the DNA-DNA hybrid reduces the DNA-RNA hybrid stability, and causes the transcription complex disso
33、ciated. 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,termination,termination,真核生物的转录过程The Process of Transcription in Eukaryote,第三节,真核生物的转录过程第三节,Science family,Roger D. Kornberg （2006）(left 1),Arthur Kornberg
34、（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 termination（终止）. The prokaryotic RNA-pol can bind to the DNA template directly in the transcriptio
35、n 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 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
36、 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 t,RNA-pol of eukaryotes,Amanitin(鹅膏蕈碱)is a specific inhibitor of RNA-pol.,RNA-polIIIIII products45S rRN,Two large subunits (similar to subunit) and 12-15 smaller subunits.The
37、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 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
38、any subunit similar to factor. Therefore, the initiation of transcription is mediated by other proteins.,each of RNA pol is a multiple-subunit protein and its structure is more complex than those in prokaryotic cell.,Two large subunits (similar to,3.2 Transcription initiation needs promoter（启动子） ，up
39、stream regulatory regions（上游调控元件） ，RNA pol and transcription factor （转录因子）.,（1）the core promoter element of Transcription initiation upstream section,The 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
40、initiation,cis-acting elements include promoter, upstream promoter elements (or promoter-proximal elements, 启动子上游元件) and enhancer（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）。,cis-acting
41、 elements include pr,TATA box,Similar to -10 region（70recognition）,TATA box Similar to -10 regi,Cis-acting element,Cis-acting element,RNA pol : hnRNA,RNA pol : hnRNA,RNA 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 Transc
42、ript products:,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 regions ( ICR,内部控制区).,RNA Pol IIITranscript products,RNA-pol does not bind the promoter directly. RNA-pol II associates
43、with six transcription factors, TFII A - H. The trans-acting factors （反式作用因子）are the proteins（蛋白质） 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 transcript
44、ional 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,参与RNA-pol转录的TF,型基因中的四类转录因子,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
45、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 prote,TF for eukaryotic transcription,TF for eukaryotic transcriptio,（3） Pre-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,
46、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 complex (PI,Pre-initiation complex (PIC),Pre-initiation complex (PIC)TB,RNA的生物合成课件,TF II H is of protein
47、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） of RNA-pol,TF II H is of protein kinase,PI
48、C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 inducible factors and upstream factors ,可诱导因子和上游因子） bind together to form active and specific complex which can bi
49、nd 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-act,RNA的生物合成课件,Promoter and regulatory proteins,Promoter and regulatory prote,3.3 Eukaryotic transcription elongation process of transcription and transl
50、ation 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）. As RNA-pol move, it may reach nucleosome（核小体）. You can observe the movement and degradation of nucleosome.,3.3
