現(xiàn)代分子生物學-07RNA的合成.ppt

1、7 RNA的合成 (RNA transcription), 基因表達的第一步, 以D. S. DNA中的一條單鏈作為轉錄的模板, 在依賴DNA的RNA聚合酶的作用下, 模板單鏈 DNA的極性方向為3 5, 而非模板單鏈 DNA的極性方向與RNA鏈相同，均為5 3.,DNA,(書寫DNA序列時，僅寫非模板序列，可不注明極性方向),3-TACTCAT-5,RNA 5-AUGAGUA-3,5-ATGAGTA-3,Non-template (sense strand),template (antisense strand),若 干 基 本 概 念, 某一基因只以一條單鏈DNA 為模板進行轉錄（不對稱轉

2、錄）, RNA的轉錄包括promotion, elongation, termination 三過程, 從啟動子（promoter）到終止子（terminator）稱為 轉錄單位 （transcriptional unit）, 原核生物中的轉錄單位多為 polycistron in operon, 轉錄原點記為+1，其上游記為負值，下游記為正值,真核生物中的轉錄單位多為monocistron, No operon,7.1.原核生物RNA的結構及種類,7.2 RNA酶促合成的特點,Rifampin對RNApol 的抑制表現(xiàn), 當I, E site被pppXpYpZ(3NMP)填充， Rif失去抑

3、制效應,Rifampin是RNA合成起始的抑制劑,7.3 RNA polymerase in prok.,RNA polymerase in prok.,Core Enzyme,Holo Enzyme,因子；, 重復使用(Reusable), 使 Holoenzyme識別Sextama Box, 與模板鏈結合, 修飾 RNApol 構型，,降低全酶與DNA的非專一性結合力（107/mol）,增強全酶與R, B site的專一性結合力 (1014/mol),導致RNA鏈的延伸緩慢,因子；, 促使RNApol與DNA 模板鏈結合, 位于前端的因子使雙鏈解鏈為單鏈, 位于尾端的因子使單鏈新聚合為雙鏈

4、,因子；, 促進RNA pol + NTP RNA elongation, 完成 NMP之間的磷酸脂鍵的連接, Editing, 與 Rho()因子競爭RNA 3-end, 構成Holo Enzyme后，因子含有 兩個位點,對 NTP 非專一性結合, 因子；, 強堿性亞基, 促使RNA與 非模板鏈（sense strand）結合, 受K酶抑制,Holo Enzyme 含有五個功能位點, sense strand DNA binding point( ), DNA/RNA hybrid site(), D. S.DNA unwinding point(), D. S.DNA rewinding

5、point(), factor point,7.4. RNA Transcription promotion in prokaryots,7.4.1. Promoter 的結構與功能 ( Prok. E.coli ),a) promoter 由兩個重要部分組成, -70 -40 CAPcAMP binding site，, -35 -10 RNApol. binding site,基因表達調控的正控制位點,Promoter region identified by DNAase method,RNA polymerase (no NTP),DNase digestion DNA,+,disso

6、lve,Promoter region including,-35 (R),-10 (B),+1 (I),RNA,b) CAP-cAMP binding site, siteI + CAP-cAMP,cooperative effect,提高siteII 的結合效率,Site II + CAP-cAMP 復合體 促使Sextama Box 附近GC島區(qū)的雙螺旋結構穩(wěn)定性降低， Pribnow Box 的解鏈溫度降低，利于轉錄啟動,RNApol,c) Pribnow Box 的突變與遺傳效應,-C17 TATAAT,G,-pRE AAGTAT,C,A,C,14/16 down mutation,

7、2/16 up mutation, Pribnow Box 中A/TT/A, 改變了RNApol. 與模板鏈的結合效率 轉錄效率上升（或下降）,l Sextama Box 與Pribnow Box 間距17bp, 有利于RNApol啟動,l Sextama Box or Pribnow Box mut.,間距趨近于17 bp，up mutation,間距遠離于17 bp，down mutation,17bp的間距較17bp的序列對轉錄更為重要,Sextama Box and Pribnow Box mut., 轉錄率下降100X, 轉錄率下降1000X,7.5 RNA的酶促合成,After 2

8、 Nt,NusA protein ;, 69 Kd, acid protein, RNApol-attaching factor, Impel RNApol。 pausing in terminator for 1-15 and waiting for Rho factor to stop transcription of RNA,(N protein utilization substance),7.6. RNA transcription promotion in Eukaryotes,7.6.1. Biological state before transcriptional start

9、ing,l 細胞膜,l Transcriptional factor (TF, trans factor) RNApol Cis-factor,l 染色體狀態(tài)的調整 核小體 包裝的解體 H3 protein outer DNaseIS,外界信息的感知與傳遞,細胞信號傳遞,complex,細胞核,l 基本水平轉錄的 cis-factor ( promoter or basic factor) Core promoter (Cap site, TATA box 必需因子) UPE (Upstream Promoter Element) (CAAT box, GC box) For housekee

10、ping gene (constitutive expression),7.6.2. 與轉錄啟動相關的cis-factors,l 特異誘導高效表達的cis-factor Enhancer For luxury gene (inducible expression),Promoter for basic transcription,l Cap site ; initiation point (+1) Capping m7GpppA/G-70 -AUG-(in mRNA),Promoter for basic transcription,l TATA box / Hogness box / Gol

11、dberg-Hogness box (-30) Rich AT and rich GC flanked rich GC-T A T A A (T) A A (T)-rich GC 82 97 93 85 63 (37) 83 50 (37),Promoter for basic transcription,l GC island (C no methylated) and CAAT box (UPE) (-70) GCGC-GGC(T)CAATCT- Response to effect of transcription Range 30 bp,l Enhancer,Enhance expre

12、ssion Basic expression,Position not be fixed isolated region,Bi-directional element Mono-directional element, Enhancer與Promoter的比較,Promoter clearance,Transcription starting,complete TIC,7.7. Transcription termination,7.7.1. 概念,7.7.2. Terminator 種類,基因末端終止子(terminator) 基因內部終止子（attenuator） Rho-independ

13、ent terminator / Rho-dependent terminator,Difficult to identify the primary transcript,l RNApol II leave from terminator,In prokaryotes,In Eukaryotes,l RNA released from Triplex (RNA, DNA, RNApol II),l different from discontinue / read-through,because pre-mRNA processing,7.7.3. Rho-independent termi

14、nator (simple terminator),7.7.4.Rho-dependent terminator,7.8. Pre-RNA processing in Eukaryotes,7.8.1. 概念;,pre-RNA,capping tailing splicing methylation editing,生物學意義;,mature RNA, prevent pre-RNA from digested by RNase,7.8.2. pre-RNA capping,l Cytoplasm Polyhedron Virus (CPV) Jan Furuich (1974),Cap-0

15、m7GpppXpYp- (共有) Cap-1 m7GpppXmpYp- Cap-2 m7GpppXmpYmp-, After 30 Nt tramscripted, pre-RNA capping start,5 pppXpY-(30 Nt)-3 (pre-RNA),pi,ppXpY-,mRNA capping proceeding,GTP,SAM,ppXpYp-,GpppXpYp-,SAH,ppi,Gp,m7,-(with Cap-0),SAM,SAM,m7GpppXmpYp-,m7GpppXmpYmp-,SAM; S - Adenosyl - L - methionine SAH; S -

16、 Adenosyl - L - homocysteine,SAH,(with Cap-1),SAH,(with Cap-2),7.8.3. pre-RNA tailing, 概念 A poly(A) tail (50-200) be added at -20 Nt tailing signal (AAUAAA) from 3-end of Pre-RNA,特異 內切酶識別AAUAAA及隨后的GUGUGUG并在其間酶切，在3端聚合50-200poly(A),7.8.4. RNA internal methylation,m5C,m6A,CH3,H3C,常見tRNA中的修飾核苷酸,RNA splicing models