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第17章RNA的合成(新)


这是位于甘肃省酒泉市阿克塞哈萨克族自治县县城西约50公里红柳 沟的柱廊状宫殿式丹霞地貌局部(4月20日摄)。 甘肃省酒泉市阿克塞哈萨克族自治县红柳沟的丹霞地貌呈南北走 向、全长约6公里,处在阿尔金山主峰脚下,以柱廊、宫殿等形状为 主,壮观美丽,堪称鬼斧神工。 这段丹霞地貌过去曾长期不被外界了解,2007年经有关丹霞地貌 专家考察,确定其为柱廊状宫殿式丹霞地貌。

壮美的南极

中部リヨンで18日、市内を流れるローヌ川に巨大なネ ズミの模型が出現。仰向けになって横たわる姿が何と もかわいらしいが、制作した建築家によると、洪水の 危険性について人々に理解してもらうのが目的という

アフリカチビネズミ

世界最贵的老鼠10-15万瑞士法郎

第十七章RNA的生物合成

Key Terms
? ? ? ? ? ? ? ? ? ? ? transcription RNA polymerase promoter sites transcription factor footprinting consensus sequence sigma subunit transcription bubble rho (r) protein TATA box enhancer

pre-mRNA 5 cap poly(A) tail RNA editing RNA splicing spliceosome small nuclear RNA (snRNA) alternative splicing catalytic RNA self-splicing III. Synthesizing

RNA的合成过程
(RNA)n +NTP → (RNA)n +1 + PPi RNA合成需要; 双链DNA 活化前提(NTP) 镁离子或锰离子 酶类

包括起始,延伸和终止三个部分

? 识别阶段;酶与启动子的结合(全酶)DNA 链的局部打开. ? 转录的起始阶段; DNA摸板和RNA碱基配对 ? RNA链的延长; 核心酶向前移动,RNA链延 长. ? 转录的终止;酶到达基因转录终点. ? 新合成的RNA 和 RNA 聚合酶从DNA上脱落.

Transcription Is Catalyzed by RNA Polymerase
? We begin our consideration of transcription by examining the process in bacteria such as E. coli. RNA polymerase from E. coli is a very large (~400 kd) and complex enzyme consisting of four kinds of subunits .The subunit composition of the entire enzyme, called the holoenzyme, is a 2 b b s. The s subunit helps find a promoter site where transcription begins, participates in the initiation of RNA synthesis, and then dissociates from the rest of the enzyme. RNA polymerase without this subunit (a 2 b b ) is called the core enzyme. The core enzyme contains the catalytic site. This catalytic site resembles that of DNA polymerase in that it includes two metal ions in its active form. One metal ion remains bound to the enzyme, whereas the other appears to come in with the nucleoside triphosphate and leave with the pyrophosphate. Three conserved aspartate residues of the enzyme participate in binding these metal ions. Note that the overall structures of DNA polymerase and RNA polymerase are quite different; their

一 RNA聚合酶
☆1960-1961年发现,全酶分子量465000,有五种 亚基 组成,含有两个锌原子 ☆全酶中无希格码亚基的酶称为核心酶,核心酶只能 使已开始的RNA链延长,不具有起始合成RNA的能力 ☆希格码亚基称为起始亚基 ☆ RNA聚合酶的作用是合成一条与被转录的DNA互

补的反平行的RNA链。RNA以5?→3?方向合成,而
RNA聚合酶沿着模板链3?→5?方向移动。

RNA Polymerase Structures. The three-dimensional structures of RNA polymerases from a prokaryote(Thermus aquaticus) and a eukaryote (Saccharoromyces cerevisiae). The two largest subunits for each structureare shown in dark red and dark blue. The similarity of these structures reveals that these enzymes have the same evolutionary origin and have many mechanistic features in common.

RNA Chains Are Formed de Novo and Grow in the 5ˊ -to-3 ˊ Direction

★RNA聚合酶的组成



二 真核生物RNA聚合酶
结构含有8-14个亚基 RNA聚合酶 I: 存在于核仁, 转录 28S,18S,5.8S rRNA. RNA聚合酶II: 存在于核质, 转录mRNA RNA聚合酶III: 存在于核质, 转录tRNA和 5SrRNA

Eukaryotic RNA polymerases
Type Location Cellular transcripts amanitin Effects of a-

I Nucleolus 18S, 5.8S, and 28S rRNA Insensitive II Nucleoplasm mRNA precursors and snRNA Strongly inhibited III Nucleoplasm tRNA and 5S rRNA Inhibited by high concentrations

RNA in Eukaryotic Cells Is Synthesized by Three Types of RNA Polymerase
? In prokaryotes, RNA is synthesized by a single kind of polymerase. In contrast, the nucleus of a eukaryote contains threetypes of RNA polymerase differing in template specificity, location in the nucleus, and susceptibility to inhibitors (Table28.2). All these polymerases are large proteins, containing from 8 to 14 subunits and having a total molecular massgreater than 500 kd. RNA polymerase I is located in nucleoli, where it transcribes the tandem array of genes for 18S,5.8S, and 28S ribosomal RNA (Section 29.3.1). The other ribosomal RNA molecule (5S rRNA, Section 29.3.1) and allthe transfer RNA molecules (Section 29.1.2) are synthesized by RNA polymerase III, which is located in the nucleoplasmrather than in nucleoli. RNA polymerase II, which also is located in the nucleoplasm, synthesizes the precursors of messenger RNA as well as several small RNA molecules, such as those of the splicing apparatus (Section 28.3.5). Although all eukaryotic RNA polymerases are homologous to one another and to prokaryotic RNA polymerase, RNApolymerase II contains a unique carboxyl-terminal domain on the 220-kd subunit; this domain is unusual because it contains multiple repeats of a YSPTSPS consensus sequence. The activities of RNA polymerase II are regulated byphosphorylation on the serine and threonine residues of the carboxyl-terminal domain. Another major distinction among the polymerases lies in their responses to the toxin a -amanitin, a cyclic octapeptide that contains several modified

?

? amino acids.a-Amanitin is produced by the poisonous mushroom Amanita phalloides, which is also called the death cup or the destroying angel (Figure 28.16). More than a hundred deaths result worldwide each year from the ingestion of poisonous mushrooms. a-Amanitin binds very tightly (K d = 10 nM) to RNA polymerase II and thereby blocks the elongation phase of RNA synthesis. Higher concentrations of a-amanitin (1 mM) inhibit polymerase III, whereas polymerase I is insensitive to this toxin. This pattern of sensitivity is highly conserved throughout the animal and plant kingdoms.

Transcription and Translation. These two processes are closely coupled in prokaryotes, whereas they are spacially and temporally separate in eukaryotes. (A) In prokaryotes, the primary transcript serves as mRNA and is used immediately as the template for protein synthesis. (B) In eukaryotes, mRNA precursors are processed and spliced inthe nucleus before being transported to the cytosol for translation into protein. [After J. Darnell, H. Lodish, and D.Baltimore. Molecular Cell Biology, 2d ed. (Scientific American Books, 1990), p. 230.]

★转录泡

三 摸板链 非摸板链

负链 无意义链 正链 有意义链

DNA Unwinding. RNA polymerase unwinds about 17 base pairs of template DNA.

Transcription Bubble. A schematic representation of a transcription bubble in the elongation of an RNA transcript. Duplex DNA is unwound at the forward end of RNA polymerase and rewound at its rear end. The RNA-DNA hybrid rotates during elongation.

★摸板链转录的RNA链与非摸板链碱基序列相同, 只是用U代替了T ★ In contrast, the coding strand of DNA has the same sequence as that of the RNA transcript except for thymine (T) in place of uracil (U).

四 RNA聚合酶与启动子的识别
启动子(Promoter)

? The first nucleotide (the start site) of a transcribed DNA sequence is denoted as +1 and the second one as +2; the nucleotide preceding the start site is denoted as -1. These designations refer to the coding strand of DNA. Recall that the sequence of the template strand of DNA is the complement of that of the RNA transcript (see Figure 5.26). In contrast, the coding strand of DNA has the same sequence as that of the RNA transcript except for thymine (T) in place of uracil (U). The coding strand is also known as the sense (+) strand, and the template strand as the antisense (-) strand.

Prokaryotic Promoter Sequences. A comparison of five sequences from prokaryotic promoters reveals arecurring sequence of TATAAT centered on position -10. The -10 consensus sequence (in red) was deduced(推断) from a largenumber of promoter sequences. The sequences are from the (A) lac, (B) gal, and (C) trp operons of E. coli; (D) from lphage; and (E) from F C174 phage. III. Synthesizing

启动子
在RNA合成中用作模板的链称模板链或负()链。与模板链互补的DNA链称为非模板链或正 (+)链。非模板链与转录的RNA在碱基序列上 是一致的,只是DNA中用T代替了U。非模板链 有时又称为编码链,它在转录和蛋白质合成中没 有直接功能。RNA合成中转录起始点标上+1,这 个位点前面的碱基对标以负数,它们是不被转录 的,而起始之后的碱基序列标以正数。这里不设 0数。5?端方向称上游(up stream),3?端方向称 下游(downstream),根据习惯是指非模板链中 的序列。

? 实验证明,启动子序列中某些核苷酸是保守的 ,即在大多数情况下它们都会出现,称为共有 序列。原核与真核生物启动子通常含有两个共 有序列。原核生物共有序列出现在-10和-35所 以称-10区和-35区。真核生物共有序列出现在 -25和-75,分别称为-25区(TATA box或 Hoghess box)和-75区(CAAT box)。紧 邻转录起始点的共有序列是一个A/T富含区, 它使DNA双链容易局部解链。

五 起始
1.δ亚基与核心酶结合形成RNA聚合酶全酶。 2.全酶以静电作用和启动子上游非特异性DNA 结合。 3.RNA聚合酶全酶沿DNA上下游动,包括共有序列在内 与启动子特异性结合,全酶结合后引起DNA片段部分解 链伸展开约11bp(从-9~+2) 4.酶与ATP或GTP结合,结合的三磷酸核苷酸形成转录 物的5?末端,刚结合上的三磷酸核苷3?→OH与下一个 核苷酸生成磷酸酯键。所以真核和原核生物的转录作用 以A或G开始。 5.当全酶继续向下游移动,约有4个核苷酸与ATP(GTP )进行聚合。δ亚基从全酶上解离,核心酶继续合成。 新RNA链生长方向是5?→3?,新合成的RNA链与被转 录的DNA链互补,反平行。

Sigma Subunits of RNA Polymerase Recognize Promoter

六 RNA链的延长
RNA链的延长是5′→3′方向,按碱基互补原则依次连接 核苷酸,每秒合成最大速度为50个核苷酸,转录由启动子 至终止子 RNA聚合酶全酶识别起始位点,合成几个磷酸二酯键,希 格玛亚基释出核心酶继续催化,酶沿着DNA摸板3′ → 5′移 动,前面的DNA双股渐打开,转录过的区域又形成双股 DNA。 转录中DNA双股中只有一股为摸板链 5‘端是pppG 或 pppA 从头合成,第二个核苷酸就与 ATP(或GTP)的3?-OH形成3?,5?-磷酸二酯键继而参入 RNA链。在参入核苷酸的过程中重复形成3?,5?-磷酸二酯 键构成转录的延长阶段,此阶段RNA链不断延伸。 RNA聚合酶后面的DNA恢复双螺旋结构,当到达DNA的一 个特殊位点时,转录即停止。 RNA聚合酶无校对功能故转录出现的误差是复制的10万倍 。由于一个基因的转录产物很多因而可以耐受如此大的误 差。

七 RNA链的终止
终止包括:停止RNA链的延长 新生RNA链的释放 RNA聚合酶从DNA上释放 原核生物的终止子 ? 原核生物的终止子在终止点之前均有一 个回文结构,其产生的RNA可形成发卡 结构。在终点前还有一系列U,回文对称 区通常有一段富含G-C的序列。

Termination Signal. A termination signal found at the 3 end of an mRNA transcript consists of a series of bases that form a stable stem-loop structure and a series of U residues.

大肠杆菌的两类终止子
? 依赖于rho(ρ)终止子;必须在rho(ρ)存在下发 生终止。此类终止子其回文结构不富含G-C区, 回文结构之后也无寡聚U。 ? 不依赖于rho(ρ)终止子(简单终止子);此类终 止子除能形成发卡结构外,在终点有6个U核 苷酸,回文对称去有一段富含G-C序列。 rho(ρ); 分质量为46000的蛋白质,通常以6聚 体的形式存在。在有RNA存在时它能水解三磷 酸腺苷,rho(ρ)可沿RNA链移动。有rho(ρ)时 合成RNA链短。无rho(ρ)时合成RNA链长。

E.coli的转录终止有两种机制
★不依赖rho因子的终止机制。 在富含A/T的DNA基础上,前段还富含G/C的节段 ,富含G/C的RNA转录物自身回折形成一种发夹结构 。发夹结构的末端还有一连续U碱基序列可与模板链A 碱基序列互补,当RNA转录物形成发夹结构,迫使聚 合酶暂时停止作用。 ★依赖rho(ρ蛋白) 因子的终止机制。 在无A/T和G/C富含节段发生的,需rho参加,rho 因子是由相同的6个亚基组成的寡聚蛋白质。 rho蛋白 只有在单链RNA存在的情况下水解ATP与新生单链 RNA结合。 rho蛋白的ATP酶活性使rho蛋白沿新生 RNA链转录泡单方向移动。将RNA-DNA分开,终止 转录。

Effect of rho Protein On the Size of RNA Transcripts.

Mechanism For the Termination of Transcription by r Protein. This protein is an ATP-dependent helicase that binds the nascent RNA chain and pulls it away from RNA polymerase and the DNA template.

八 RNA生物合成的抑制剂
? 嘌呤和嘧啶的类似物
人工合成的碱基类似物

? DNA摸板功能的抑制物
烷化物 放线菌素 嵌入染料

? RNA聚合酶的抑制物
利福酶素 利链菌素

九 RNA转录后加工
? 转录后的RNA最初是以一种完全没有 活性的形式存在,称为初级转录物( primarytranscript)。这种初级转录 物需要进行转录后加工,使之转变成 有活性和成熟的RNA。转录后的加工 包含3种共有的分子变化:1从初级转 录物上移去核苷酸;2给转录物添加上 核苷酸;3转录物中特殊碱基的修饰。

? 原核生物的rRNA 的加工 ? 真核生物的rRNA 的加工 ? 原核生物的tRNA的加工 ? 原核生物的mRNA的 加工 ?真核生物的mRNA的 加工

核糖体RNA(rRNA)前体和转移RNA( tRNA)前体的加工
? 1.原核生物的rRNA前体沉降系数是30S含有一个拷 贝的5S,16S,23S rRNA和几个tRNA前体。 ? 2.真核生物的rRNA前体沉降系数为35~47S,含有 一个拷贝的5.8S,18S和28SrRNA。第四个真核 rRNA即5SRNA只有一个单独的前体。经RNA酶切割 前导序列成为成熟的核糖体RNA。 ? 3.原核和真核的tRNA的转录过程也是先合成大的前 体。大多数真核tRNA前体在反密码子附近有一个内含 子,并含有前导序列。经专一的核酸酶连续作用将它 们切除,用CCA-OH替代3?端UU后而成为成熟的 tRNA。

Primary Transcript. Cleavage of this transcript produces 5S, 16S, and 23S rRNA molecules and a tRNA molecule. Spacer regions are shown in yellow.

前导序列

酵母移去14个核苷酸

Transfer RNA Precursor Processing. The conversion of a yeast tRNA precursor into a mature tRNA requires the removal of a 14-nucleotide intron (yellow), the cleavage of a 5 leader (green), and the removal of UU and the attachment of CCA at the 3 end (red). In addition, several bases are modified.

★核酸酶
? 核酸酶P是专一的核糖核酸酶。催化大多数 tRNA 前提产生分子的5′端(切除前导序列 形成pG)。由-RNA分子(377核苷酸)和 一个蛋白质分子组成,保持完整的酶活性两者 都需要。但催化亚基是RNA而不是蛋白质,蛋 白质只起保持RNA正确折叠和最大的催化活性 。核酸酶P(ribozyme)是一种具有工具酶一 样催化活性的核酸。

mRNA的加工
1.原核mRNA不进行加工,转录和翻译偶联同时进行。 2.真核生物mRNA初级转录物要经过剪接外显子、戴帽、多聚腺 苷酸化等加工过程。 a. 外显子剪接 许多真核基因是以不连续基因或割裂基因出现。有些序列戴有遗 传信息,为RNA节段编码,称为外显子,有些序列不为RNA编 码,但含有某些调控成分,这种插入的序列称为内含子。内含 子将一个外显子与另一个外显子分隔开,mRNA前体中存在内 含子和外显子的相应序列。加工过程主要是切除内含子后连接 外显子。它是由核内小RNA(snRNA)和核内小核糖核糖白( snRNP)聚集形成专一性很差的复合物,称剪接体。剪接体通 过自身的snRNA和转录物之间碱基互补来识别剪接位点。

b. mRNA5?端戴帽和3?端加尾
真核mRNA需要进行两种修饰。在5?端加 一甲基化帽子和在 3?端加上多聚腺苷酸( poly A)尾。转录进行时5?末端的修饰作 用就立即开始,构成共转录加工加帽反应 分四步进行。真核mRNA合成后需要在3?OH端加上一个poly A尾巴。核酸内切酶从 mRNA前体的高度保守的AAUAAA切割信 号下游10~30个核苷酸的位点进行酶切, 然后在聚腺苷酸酶催化下加上poly A。 poly A可保护mRNA的3?-末端不会被核酸 酶降解,并有助于mRNA从细胞投向胞浆 转移。

Capping the 5 End. Caps at the 5 end of eukaryotic mRNA include 7-methylguanylate (red) attached by a triphosphate linkage to the ribose at the 5 end. None of the riboses are methylated in cap 0, one is methylated in cap 1, and both are methylated in cap 2.

m7G-5′-PPP-5′-Nm

帽子结构常被甲基化

Cap 0 Cap 1 Cap 2 m7G-5′-PPP-5′-Nm

帽子的功能 ★使mRNA免遭核酸酶的破坏 ★能使mRNA与核糖体小亚基结合,开始合成 蛋白质 ★被蛋白质合成的起始因子识别,促进蛋白质 合成

Polyadenylation of a Primary Transcript. A specific endonuclease cleaves the RNA downstream of AAUAAA. Poly(A) polymerase then adds about 250 adenylate residues.

绝大多数真核生物mRNA具Poly A尾巴 功能

?mRNA进入细胞质所必须? ? 提高mRNA在细胞质中的稳定性
真核生物mRNA的拼接 真核生物mRNA的甲基化修饰

cDNA

真核生物mRNA的加工
? 转录含有外显子和内含子的mRNA的前 体(hnRNA) ? 切除内含子 ? 5‘端帽子结构和3’端多聚A的结构

dimensional structures of RNA polymerases from a prokaryote (Thermus aquaticus) and a eukaryote (Saccharoromyces cerevisiae). The two largest subunits for each structure are shown in dark red and dark blue. The similarity of these structures reveals that these enzymes have the same evolutionary origin and have many mechanistic features in common

.

Summary
? ? Transcription Is Catalyzed by RNA Polymerase All cellular RNA molecules are synthesized by RNA polymerases according to instructions given by DNA templates.The activated monomer substrates are ribonucleoside triphosphates. The direction of RNA synthesis is 5 3 , as in DNA synthesis. RNA polymerases, unlike DNA polymerases, do not need a primer and do not possess proofreading nuclease activity.RNA polymerase in E. coli is a multisubunit enzyme. The subunit composition of the ~500-kd holoenzyme is a 2 b b s and that of the core enzyme is a 2 b b . Transcription is initiated at promoter sites consisting of two sequences, one centered near -10 and the other near -35; that is, 10 and 35 nucleotides away from the start site in the 5 (upstream) direction. The consensus sequence of the -10 region is TATAAT. The s subunit enables the holoenzyme to recognize promoter sites. When the growth temperature is raised, E. coli expresses a special s that selectively binds the distinctive promoter of heatshock genes. RNA polymerase must unwind the template double helix for transcription to take place.Unwinding exposes some 17 bases on the template strand and sets the stage for the formation of the first phosphodiester bond. RNA chains usually start with pppG or pppA. The s subunit dissociates from the holoenzyme after the initiation of the new chain. Elongation takes place at transcription bubbles that move along the DNA template at a rate of about 50 nucleotides per second. The nascent RNA chain contains stop signals that end transcription. One stop signal is an RNA hairpin, which is followed by several U residues. A different stop signal is read by the rho protein, an ATPase. In E. coli, precursors of transfer RNA and ribosomal RNA are cleaved and chemically modified after transcription, whereas mRNA is used unchanged as a template for protein synthesis.

Eukaryotic Transcription and Translation Are Separated in Space and Time
RNA synthesis in eukaryotes takes place in the nucleus, whereas protein synthesis takes place in the cytoplasm. There are three types of RNA polymerase in the nucleus: RNA polymerase I makes ribosomal RNA precursors, II makes messenger RNA precursors, and III makes transfer RNA precursors. Eukaryotic promoters are complex, being composed of several different elements. Promoters for RNA polymerase II are located on the 5 side of the start site for transcription. Each consists of a TATA box centered between -30 and -100 and additional upstream sequences. They are recognized by proteins called transcription factors rather than by RNA polymerase II. The saddle-shaped TATA-boxbinding protein unwinds and sharply bends DNA at TATA-box sequences and serves as a focal point for the assembly of transcription complexes. The TATA-box-binding protein initiates the assembly of the active transcription complex. The activity of many promoters is greatly increased by enhancer sequences that have no promoter activity of their own. Enhancer sequences can act over distances of several kilobases, and they can be located either upstream or downstreamof a gene.

The Transcription Products of All Three Eukaryotic Polymerases Are Processed
? The 5 ends of mRNA precursors become capped and methylated in the course of transcription. A 3 poly(A) tail is added to most mRNA precursors after the nascent chain has been cleaved by an endonuclease. RNA editing processes alter the nucleotide sequence of some mRNAs, such as the one for apolipoprotein B. The splicing of mRNA precursors is carried out by spliceosomes, which consist of small nuclear ribonucleoprotein particles (snRNPs). Splice sites in mRNA precursors are specified by sequences at ends of introns and by branch sites near their 3 ends. The 2 -OH group of an adenosine residue in the branch site attacks the 5 splice site to form a lariat intermediate. The newly generated 3 -OH terminus of the upstream exon then attacks the 3 splice site to become joined to the downstream exon. Splicing thus consists of two transesterification reactions, with the number of phosphodiester bonds remaining constant during reactions. Small nuclear RNAs in spliceosomes catalyze the splicing of mRNA precursors. In particular, U2 and U6 snRNAs form the active centers of spliceosomes.

The Discovery of Catalytic RNA Was Revealing with Regard to Both Mechanism And Evolution ? Some RNA molecules, such as the 26S ribosomal RNA precursor from Tetrahymena, undergo self-splicing in the absence of protein. A self-modified version of this rRNA intron displays true catalytic activity. Spliceosome-catalyzed splicing may have evolved from selfsplicing. The discovery of catalytic RNA has opened new vistas in our exploration of early stages of molecular evolution.


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