1、7 RNA的合成(RNA transcription), 基因表达的第一步, 以D. S. DNA中的一条单链作为转录的模板, 在依赖DNA的RNA聚合酶的作用下, 模板单链 DNA的极性方向为3 5, 而非模板单链DNA的极性方向与RNA链相同，均为5 3.,DNA,(书写DNA序列时，仅写非模板序列，可不注明极性方向),3-TACTCAT-5,RNA 5-AUGAGUA-3,5-ATGAGTA-3,Non-template (sense strand),template (antisense strand),若 干 基 本 概 念, 某一基因只以一条单链DNA 为模板进行转录（不对称转录）
2、, RNA的转录包括promotion, elongation, termination 三过程, 从启动子（promoter）到终止子（terminator）称为转录单位 （transcriptional unit）, 原核生物中的转录单位多为 polycistron in operon, 转录原点记为+1，其上游记为负值，下游记为正值,真核生物中的转录单位多为monocistron, No operon,7.1. 原核生物RNA的结构及种类,7.2 RNA酶促合成的特点,Rifampin对RNApol 的抑制表现, 当I, E site被pppXpYpZ(3NMP)填充，Rif失去抑制效应
3、,Rifampin是RNA合成起始的抑制剂,7.3 RNA polymerase in prok.,RNA polymerase in prok.,Core Enzyme,Holo Enzyme,因子；, 重复使用(Reusable), 使 Holoenzyme识别Sextama Box, 与模板链结合, 修饰 RNApol 构型，,降低全酶与DNA的非专一性结合力（107/mol）,增强全酶与R, B site的专一性结合力 (1014/mol),导致RNA链的延伸缓慢,因子；, 促使RNApol与DNA 模板链结合, 位于前端的因子使双链解链为单链, 位于尾端的因子使单链新聚合为双链,因子
4、；, 促进RNA pol + NTP RNA elongation, 完成 NMP之间的磷酸脂键的连接, Editing, 与 Rho()因子竞争RNA 3-end, 构成Holo Enzyme后，因子含有 两个位点,对 NTP 非专一性结合, 因子；, 强碱性亚基, 促使RNA与非模板链（sense strand）结合, 受K酶抑制,Holo Enzyme 含有五个功能位点, sense strand DNA binding point( ), DNA/RNA hybrid site(), D. S.DNA unwinding point(), D. S.DNA rewinding poin
5、t(), factor point,7.4. RNA Transcription promotion in prokaryots,7.4.1. Promoter 的结构与功能 ( Prok. E.coli ),a) promoter 由两个重要部分组成, -70 -40 CAPcAMP binding site，, -35 -10 RNApol. binding site,基因表达调控的正控制位点,Promoter region identified by DNAase method,RNA polymerase (no NTP),DNase digestion DNA,+,dissolve,
6、Promoter region including,-35 (R),-10 (B),+1 (I),RNA,b) CAP-cAMP binding site, siteI + CAP-cAMP,cooperative effect,提高siteII 的结合效率,Site II + CAP-cAMP 复合体促使Sextama Box 附近GC岛区的双螺旋结构稳定性降低，Pribnow Box 的解链温度降低，利于转录启动,RNApol,c) Pribnow Box 的突变与遗传效应,-C17 TATAAT,G,-pRE AAGTAT,C,A,C,14/16 down mutation,2/16 u
7、p mutation, Pribnow Box 中A/TT/A, 改变了RNApol. 与模板链的结合效率转录效率上升（或下降）,l Sextama Box 与Pribnow Box 间距17bp,有利于RNApol启动,l Sextama Box or Pribnow Box mut.,间距趋近于17 bp，up mutation,间距远离于17 bp，down mutation,17bp的间距较17bp的序列对转录更为重要,Sextama Box and Pribnow Box mut., 转录率下降100X, 转录率下降1000X,7.5 RNA的酶促合成,After 2 Nt,NusA
8、 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 starting,l 细胞
9、膜,l Transcriptional factor (TF, trans factor)RNApolCis-factor,l 染色体状态的调整核小体 包装的解体H3 protein outerDNaseIS,外界信息的感知与传递,细胞信号传递,complex,细胞核,l 基本水平转录的 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 housekeeping gene (cons
10、titutive expression),7.6.2. 与转录启动相关的cis-factors,l 特异诱导高效表达的cis-factorEnhancer 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 / Goldberg-Hogness box
11、 (-30)Rich AT and rich GC flankedrich GC-T A T A A (T) A A (T)-rich GC82 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 transcriptionRange 30 bp,l Enhancer,Enhance expression Basic expression,
12、Position not be fixed isolated region,Bi-directional element Mono-directional element, Enhancer与Promoter的比较,Promoter clearance,Transcription starting,complete TIC,7.7. Transcription termination,7.7.1. 概念,7.7.2. Terminator 种类,基因末端终止子(terminator) 基因内部终止子（attenuator）Rho-independent terminator / Rho-dep
13、endent 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 terminator(simple terminator)
14、,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,生物学意义;,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 m7GpppXpYp- (共有) Cap-1 m
15、7GpppXmpYp- 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 - Adenosyl - L - homocyst
16、eine,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,特异 内切酶识别AAUAAA及随后的GUGUGUG并在其间酶切，在3端聚合50-200poly(A),7.8.4. RNA internal methylation,m5C,m6A,CH3,H3C,常见tRNA中的修饰核苷酸,RNA splicing models,a) group I of intron (nuclear & mitochondrial),II of intron (mitochondrial),III of intron (nuclear & chloroplast),b) Group I splicing model (tetrehymera 35s rRNA model),Junction sequence., 5-exon-U -intron-G -exon-3,高度保守，转酯反应的攻击位点,形成RNA分子内的二级结构,本章内容结束,
