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DNA Transcription

DNA Transcription. Basic features Common to DNA replication 1) Template, Unwinding and Torsion-relieving are necessary; 2) Proceed only in the 5 ′ →3 ′direction; Uncommon to DNA replication 1) No need for primers 2) NTPs instead of dNTPs; UTP instead of dTTP

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DNA Transcription

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  1. DNA Transcription • Basic features Common to DNA replication 1)Template, Unwinding and Torsion-relieving are necessary; 2)Proceed only in the 5′→3′direction; Uncommon to DNA replication 1)No need for primers 2)NTPs instead of dNTPs; UTP instead of dTTP 3)Lacking proof-reading activity( error rate is 1 in 104 or 105 nts added ) 4)Specific regions (not all DNA sequence) can be transcribed 5)To a specific gene, only one strand can be transcribed Remember some nomenclature conventions

  2. 第十三章 RNA的代谢 RNA合成与DNA合成的比较： （1）催化方向均是5‘－3‘， 延伸的机理相同： 反应受焦磷酸水解趋动，需要模板。 （2）RNA合成不需引物（自身可以独立起始合成），且无外切酶作用（即缺乏核对能力）； DNA复制是一个半保留复制，RNA合成是 全保留的（因是单链）。 （3）RNA合成起始和终止均受严格的控制，而DNA的终止无特殊的信号。

  3. Central Dogma Gene expression Transcription Translation Retro-transcription Replication Replication

  4. 第一节 依赖DNA的RNA合成 转录的概念 以DNA分子中的某一区段的一条链为模板，在RNA聚合酶的作用下合成一段RNA链。 5‘――3‘： Coding strand P324 3‘――5： Template strand

  5. Coding strand, Sense strand, Crick strand Template strand, antisense strand, Watson strand Transcription Translation

  6. 原核生物的RNA聚合酶P324 1. 亚基的生成 2. 功能：催化RNA的合成: tRNA，rRNA，mRNA 3. 抑制剂：利福平rifampicin

  7. DNA-Dependent RNA Polymerases-RNAP • Common features RNAP DNA + NTPs /Mg2+DNA + RNAs + nPPi 2nPi • Differences between DNAP and RNAP • Prokaryotic RNAP • Eukaryotic RNAPs • Viral RNAPs

  8. All RNA polymerases require: 1) DNA template: one strand is copied 2) substrate NTPs (GTP, CTP, UTP, ATP) 3) divalent cation (Mg2+) Differences Between DNAP and RNAP 1) RNAP can initiate transcription de novo (i.e. RNAP doesn’t need a primer!) 2) RNAP has no proofreading activity (error rate is 1 in 104 or 105 nts added) 3) RNAP incorporates NTPs instead of dNTPs 4)RNAP incorporates UTP instead of dTTP

  9. RNAP in Prokaryotes 1) Structure and Function All three classes of RNAs are transcribed by the same RNA polymerase In E.coli, RNAP is 465 kD complex, with2 , 1 , 1 ', 1 (holoenzyme) • Core enzyme is 2 , 1 , 1 ’ (can transcribe but it can’t find promoters) •  recognizes promoter sequences on DNA 2) Inhibitors Rifampicin(利福霉素)& Streptolydigin (利链霉素)

  10. a a2 a2b a2bb’ = core enzyme aI aII b b’ s70 s32 s60 vegetative (principal s) heat shock (for emergencies) nitrogen starvation (for emergencies) The assembly pathway of the core enzyme (the w subunit makes this more efficient) CORE ENZYME Sequence-independent, nonspecific transcription initiation + σ SUBUNIT interchangeable, promoter recognition

  11. aI aII RNAP HOLOENZYME -s70 Promoter-specific transcription initiation b b’ s70 • In the Holoenzyme: • ' binds DNA •  binds NTPs •  and  ' together make up the active site •  subunits appear to be essential for assembly and for activation of enzyme by regulatory proteins. They also bind DNA. • s recognizes promoter sequences on DNA

  12. RNAPs in Eukaryotes • RNA polymerases I, II and III transcribe rRNA, mRNA and tRNA genes, respectively

  13. 原核生物的转录过程 1、结合 起始阶段 2、解链 3、引发 4、б因子解离 延长阶段 5、核心酶移动 6、形成3‘，5‘－磷酸二酯键 7、因子识别终止信号 8、核心酶停止转录 终止阶段 9、RNA链释放出来

  14. 结 合 RNA聚合酶（ 2ββ‘б）与启动子（promoter）结合，б组别启动子部位（-35启动子部位）。 б和β‘起连接作用。 RNA聚合酶合成RNA的方向为5‘－3‘，所以从转录起始点沿RNA聚合酶运动的方向称为下游（downstream），核苷酸残基编号依次为+2，+3…，而反方向为上游（upstream），核苷酸残基编号为-1，-2…。 原核生物的转录过程

  15. 解 链 解开一小段DNA双螺旋，以便产生单链DNA转录模板。 原核生物的转录过程

  16. 引 发 第一个核苷三磷酸上去（GTP、ATP）（模板）若第一个是C，那么是PPPG（GTP） 结合上去。 б因子的作用： 识别启动子部位 识别启动部位 原核生物的转录过程

  17. Detailed Transcriptional Mechanism • Three-step process 1) Initiation 2) Elongation 3) Termination • DNA transcription in prokaryotes • DNA transcription in eukaryotes • In vitro DNA transcription

  18. Prokaryotic DNA Transcription • Initiation 1) what is promoter? 2) how to determine the promoter sequences?-DNase I footprinting 3) Consensus sequences 4) Formation of transcriptional complex • Elongation • Termination

  19. Initiation of Transcription • Binding of RNAP to Template DNA • RNA polymerase has two binding sites for NTPs • Initiation site prefers to binds ATP and GTP (most RNAs begin with a purine at 5'-end) • Elongation site binds the second incoming NTP • 3'-OH of first attacks alpha-P of second to form a new phosphoester bond (eliminating PPi) • When 6-10 unit oligonucleotide has been made, sigma subunit dissociates, completing "initiation"

  20. RNAP bound -40 to +20 Closed complex formation RNAP unwinds from -10 to +2 Open complex formation Requires high purine [NTP] Binding of 1st NTP Requires lower [NTPs] Addition of next NTPs After RNA chain is 6-10 NTPs long Dissociation of sigma Finding and binding the promoter

  21. 延 长 阶 段 4. б因子解离 5. 核心酶移动 6. 形成3‘，5‘－磷酸二酯键 核心酶，特别是其中的β亚基起3‘，5‘－磷酸二酯键的催化作用的核心酶沿模板3‘→5‘移动，RNA链5‘→3‘延长。 原核生物的转录过程

  22. Chain Elongation Core polymerase - no sigmafactor • Polymerase is pretty accurate - only about 1 error in 10,000 bases (not as accurate as DNAP III) • Even this error rate is OK, since many transcripts are made from each gene • Elongation rate is 20-50 bases per second - slower in G/C-rich regions and faster elsewhere • Topoisomerases precede and follow polymerase to relieve super coiling

  23. Spatial Organization of Transcription Elongation Complex in E. coli Science, vol. 281, p 424 (1998)

  24. Interactions between nucleic acids and the core enzyme keep RNAP processive

  25. 终 止 阶 段 7. 因子识别终止信号（不衣赖因子） 8. 核心酶不停止转录 9. RNA链释放出来

  26. Chain Termination Two mechanisms • Rho(ρ) - the termination factor protein • rho is an ATP-dependent helicase • it moves along RNA transcript, finds the "bubble", unwinds it and releases RNA chain • Specific sequences - termination sites in DNA • inverted repeat, rich in G:C, which forms a stem-loop in RNA transcript • 6-8 As in DNA coding for Us in transcript

  27. Rho-independent transcription termination (depends on DNA sequence - NOT a protein factor) Stem-loop structure

  28. Rho-independent transcription termination • RNAP pauses when it reaches a termination site. • The pause may give the hairpin structure time to fold • The fold disrupts important interactions between the RNAP and its RNA product • The U-rich RNA can dissociate from the template • The complex is now disrupted and elongation is terminated

  29. Rho-Dependent Transcription Termination (depends on a protein AND a DNA sequence) G/C -rich site RNAP slows down Rho helicase catches up Elongating complex is disrupted

  30. 第四节 真核细胞的转录作用 1、真核有三种RNA聚合酶 (P328) 原核 真核 只有一种 3种 RNA聚合酶 不受抑制 受抑制 利福平 不同步 转录和蛋白质合成 同步

  31. 如何保证转录的忠实性：(转录差错率为10-5) 1、靠模板链与新合成链之间的碱基配对 2、核心酶对底物有专一性

  32. 转录过程的选择性抑制 抗菌素放线菌素D P330 丫啶 利福平 -鹅膏覃碱

  33. RNAP II Inhibitor • Mushrooms of the genus Amanita make a toxic cyclic octapeptide called a amanitin(鹅膏蕈碱） • This mushroom tastes good but eating it is deadly! • 6 to 24 hours after eating it violent cramps and diarrhea set in • 3rd day sees a false remission • By 4th or 5th day death will occur unless a liver transplant is done • The symptoms are due to inhibition of RNAPII and manifest mainly in liver

  34. The chemical structure of α-amanitin

  35. RNAPs in Eukaryotes RNA polymerases I, II and III have structural features in common: • All 3 are big, multimeric proteins (500-700 kD) • All have 2 large subunits with sequences similar to  and ' in E.coli RNAP, so catalytic site may be conserved • All have subunit homologs of a in E. coli RNAP • However, the eukaryotic RNA polymerase does not contain any subunit similar to the E. coliσ factor. • These features are shared by RNAPs across species

  36. Differences Transcription Bacteria vs. Eukaryotes • Multiple Polymerases –at least 3 types of RNAPs • Chromatin and Nucleosomes • Unable to initiate transcription on their own -- Require Transcription Factors (TF，转录因子) • Unable to recognize Promoters on their own • Primary transcripts contain exons • The Promoters are complex. Multiple regulatory proteins can bind to the promoter. • Cis-acting elements (顺式作用元件) and Trans-acting factors(反式作用因子). Enhancer, silencer & insulator • mRNAs are mostly monocistronic (单顺反子) • Genes controlled by positive control - off unless activators are present • In eukaryotes, transcription and translation occur in separate compartments.

  37. （三）真核生物与原核生物转录的主要区别 1. 真核细胞RNApol种类较多，根据它们对α-鹅膏蕈碱的 敏感性不同分为RNA pol I 、II 、III（or A、B、C）， 它们是高度分工的，不同的RNA聚合酶负责合成不同 的RNA。

  38. 2. 真核启动子比原核启动子更复杂和更多样性，不同的 RNA聚合酶有不同的启动子。

  39. 3. 原核细胞靠RNA pol本身可识别启动子，而真核细胞 的RNApol无法识别启动子，要靠转录因子 (transcription factor,TF）识别启动子 ，有许多转录 因子。 • 转录因子的功能：调节RNA聚合酶的活性，将RNA • 聚合酶引到启动子位置。

  40. 4. 真核生物的转录受特定的顺式作用元件（cis-acting element）的影响，顺式作用元件：真核生物DNA中 与转录调控有关的核苷酸序列，包括增强子、沉默子 等。 增强子（enhaucer）：增加转录的速度。 沉默子（silencer）：降低转录的速度，沉默子也称抑 制子。

  41. 顺式作用元件并不能直接发挥作用，要与反式作用因子（trans-acting factors）相互作用来调控转录，反式作用因子是一些特殊的蛋白质因子。

  42. 5. 原核细胞基因转录的产物大多数为多顺反子mRNA，这 是由于原核转录系统中功能相关的基因共享一个启动子， 它们在转录时，以一个共同的转录单位进行转录。而真核 细胞，每一种蛋白质的基因都有自己独立的启动子，所以 真核细胞转录产物是单顺反子mRNA。

  43. A B C DNA P 转录 mRNA A B C DNA P A P B P C 转录 mRNA A B C 原核 真核

  44. 6. 原核细胞是边转录、边翻译，两个过程几乎是同时进 行的，而真核细胞转录和翻译在时间上和空间上都是 分开的，转录在细胞核，翻译在细胞质。 7. 真核细胞中DNA与组蛋白结合在一起，形成染色质， 后者进一步盘曲、折叠形成染色体，其中只有一小 部分能转录。 8. 真核细胞被转录的产物要经过非常复杂的后加工。

  45. Relative positions of GTFs and RNAP II on DNA RNAP II crab claws clamp over the DNA near the initiation site. Melting of the DNA is assisted by the TFIIH helicase (not shown) stimulated by TFIIE

  46. recruits recruits recruit recruits and stimulates Formation of pre-initiation complex TBP (TFIID) binds to the promoter DNA (TATA + Inr) TFIIB RNAP II + TFIIF TFIIE TFIIH • Helicase activity unwinds DNA near start site • Kinase activity phosphorylates CTD

  47. Elongation by RNAP II An elongation factor called TFIIS stimulates elongation for RNAP II. TFIIF also has a role in elongation. After termination of transcription the CTD is dephosphorylated and the RNAP II can re-enter a pre-initiation complex (PIC)

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