核酸转录(双语)

1、转录 (Transcription)DNA RNA proteinCentral dogma of Molecular Biology (1970)转录（transcription）： 以基因单链DNA为模板，以NTP为原料，在RNA聚合酶等酶和蛋白质因子的作用下，按照碱基互补配对原则，反向合成互补RNA单链的过程DNA segments (structural gene) are read and transcribed into single stranded sequences of RNA. The RNA moves from the nucleus into the cytopla

2、sm. Transcription 复制 转录 模板 两股链全长复制 基因单链转录 原料 dNTP NTP 酶 DNA聚合酶 RNA聚合酶 产物 DNA双链 RNA单链 配对 A - T G - C A - U T - A C G产物后加工 某些碱基甲基化 加帽、尾、剪接、编辑等 转录的模板 (Template of Transcription)结构基因(structural gene) 能转录出RNA的DNA双链区段模板链（反意义链）- waston链： DNA双链中指引转录生成RNA的单链编码链（有意义链）- crick链： DNA双链中无转录功能的单链5-G C A G T A C A

3、T G T C-3 DNA有意义链3- c g t c a t g t a c a g -5 DNA反意义链5-G C A G U A C A U G U C-3 RNAWhat relationship between RNA transcript and the two strands of DNARNA transcript is - to the template DNA strand(antisense strand), and - to the non-template DNA(sense strand)不对称转录(asymmetric transcription)： （1）基因单

4、链转录 （2）转录模板并非固定在同一单链上 RNA聚合酶 (RNA polymerase)RNA聚合酶（DDRP） 1、5- 3聚合活性 催化子链5- 3方向核糖核苷酸之间通过磷酸二酯键连接 由于能催化游离核苷酸之间通过磷酸二酯键连接，起到引导子链起始合成的作用，转录过程无需引物酶2、无外切活性Prokaryotes1、组成：四种亚基组成的五聚体2、原核生物RNA聚合酶抑制剂： 利福平、利福霉素（专一结合 亚基抑制磷酸二酯键的合成）在转录全过程中发挥作用2核心酶2 全酶 Rifampicin is an antibiotic which is most frequently used to t

5、reat tuberculosis (TB). Rifampicin targets and inactivates a bacterial chemical called RNA-polymerase. The tuberculosis bacteria use RNA-polymerase to make essential proteins and to copy their own genetic information (DNA). Without this chemical the bacteria cannot reproduce and they die. The bacter

6、ia that cause TB are difficult to treat. By using medications in combination, the bacteria can be targeted in different ways and hence treatment is more likely to be effective than a single medicine alone. In addition, using different medications make it less likely that bacteria will develop resist

7、ance to treatment.Eukaryotes 种类 I II III初级产物 45S-rRNA hnRNA 5S-rRNA前体 tRNA前体成熟产物 5.8SrRNA mRNA 5S-rRNA 18SrRNA tRNA 28SrRNA对鹅膏蕈 耐受 极敏感 中度敏感碱的反应RNA聚合酶与模板的识别 (RNA polymerase recognize template)5RNA-pol保护区 转录基因 终止点5-50 -40 -30 -20 -10 1 10 20 TTGACA TATAATpppG转录开始RNAPromoter: A position upstream of the

8、 structural gene for RNA-pol recognizing and binding, having short conserved DNA sequences which common to different gene in different species The position of the first synthesized base of the RNA is called the start site and is designated as position +1; 启动子(promoter)： 称启动基因，为RNA聚合酶与DNA模板辨认并结合的部位，含

9、保守序列Prokaryotes:-10 region(Pribnow box): -TATAAT-35 region: -TTGACA-Eukaryotes:-25 region(Hogness/TATA box): -TATA-原核转录过程 (Transcription process in Prokaryotes)（一）转录起始1. RNA聚合酶全酶在DNA双链上滑动，迅速寻找启动子，与之形成较稳定的结构，并解开DNA双链17bp2. 在RNA聚合酶作用下，与转录起始点位置互补的两个核苷酸通过磷酸二酯键形成四磷酸二核苷，形成转录起始复合物：5 RNA pol(2)-DNA-pppGpN-O

10、H 3N（二）转录延长1. 从全酶中脱落出来2. 核心酶发生变构，与模板DNA的结合变得较为松弛，核心酶沿模板滑动，使RNA链沿5 3方向不断延长 (NMP)n + NTP (NMP)n+1 + PPi3. 生成的RNA大部分伸出转录空泡外，已被转录的DNA重新形成双螺旋DNA核糖体RNARNA聚合酶（三）转录终止1. 依赖因子的转录终止 (1) 结合RNA (2) 有ATP酶和解链酶的活性2. 不依赖因子的转录终止-茎环结构的形成-AGCCCGC-GCGGGCT-TTTTTT-TCGGGCG-CGCCCGA-AAAAAA-AGCCCGC-GCGGGCU-UUUUUU-真核转录过程 (Tran

11、scription process in Eukaryotes)Initiation:Recognition between RNA-pol and DNATermination:Post-transcriptional modification顺式作用元件(cis-acting element) DNA分子上具有的可影响转录的各种组分反式作用因子(trans-acting factors) 能直接、间接辨认和结合转录上游区段DNA的蛋白质。反式作用因子中，直接或间接结合RNA聚合酶的，则称为转录因子(transcriptional factors, TF)In the field of mo

12、lecular biology, cis-acting generally means acting from the same molecule. Trans-acting generally means acting from a different molecule. In the context of transcription regulation, cis-acting elements are usually considered to be DNA sequences that, via trans-acting factors, which are usually consi

13、dered to be proteins, regulate the expression of genes. 转录起始点TATA盒CAAT盒GC盒 增强子AATAAA切离加尾 转录终止点 修饰点 外显子 翻译起始点内含子 OCT-1 参与RNA-pol转录的TF 蛋白激酶活性，使CTD磷TFHATPaseTFE解螺旋酶TFF促进RNA-pol结合及作为其他因子结合的桥梁TFB稳定TFD-DNA复合物TFA辅助TBP-DNA结合结合 TATA 盒TFD功能转录因子蛋白激酶活性，使CTD磷酸化TFHATPaseTFE解螺旋酶TFF促进RNA-pol结合及作为其他因子结合的桥梁TFB稳定TFD-D

14、NA复合物TFA辅助TBP-DNA结合结合 TATA 盒TFD功能转录因子POL-TFFABTBPTAFTATAHECTD- P转录起始前复合物(pre-initiation complex, PIC) 真核转录后修饰 (Post-transcriptional modification in Eukaryotes)Post-transcriptional modification of mRNAPost-transcriptional modification is a process in cell biology by which, in eukaryotic cells, primar

15、y transcript RNA is converted into mature RNA. 1. Addition of 5-cap2. Addition of poly A tailThe pre-mRNA processing at the 3 end of the RNA molecule involves cleavage of its 3 end and then the addition of about 200 adenine residues to form a poly(A) tail. The cleavage and adenylation reactions occu

16、r if a polyadenylation signal sequence (5- AAUAAA-3) is located near the 3 end of the pre-mRNA molecule. A GU-rich sequence is also usually present further downstream on the pre-mRNA molecule. 5-AAUAAA-5 -AAUAAA-核酸酶-GUGUGUGRNA-polAATAAA GTGTGTG转录终止的修饰点55333加尾AAAAAAA 3 mRNAWhat is the function for 5

17、end cap and 3 end polyA of the mature mRNAThe presence of 5 cap and 3 polyA can protect mRNA from being hydrolyzed by 5 and 3 exonucleases during it transports from nucleus to cytoplasm以下实验现象说明了什么? 核酸杂交实验证明，hnRNA与模板链可以完全配对，而来自hnRNA的成熟产物mRNA与模板链出现部分配对以及中间不配对的现象，说明了什么？3. mRNA splicing How the discover

18、y was made Roberts and Sharp were studying the genetic material in adenovirus, a virus causing common cold. The genome of adenovirus consists of one single long DNA molecule. Roberts and Sharps aim was to determine where in the genome different genes were located. In biochemical experiments it was s

19、hown that one end of an adenovirus messenger RNA did not behave as expected. To determine where this segment was located on the long DNA molecule, they used electron microscopy. They surprisingly found that a single RNA molecule corresponded to no less than four well-separated segments in the DNA mo

20、lecule. Roberts and Sharp came to the conclusion that the genetic information in the gene was discontinuously organized in the genome. This knowledge has radically changed our view on how the genetic material has developed during the course of evolution. It has long been considered likely that evolu

21、tion takes place as the result of the accumulation of minor alterations in the genetic material (mutations) resulting in a gradual change. As a consequence of the discovery that genes are often split, it seems likely that higher organisms in addition to undergoing mutations may utilize another mecha

22、nism to speed up evolution: rearrangement of gene segments to new functional units. 断裂基因(split gene)： 真核生物的结构基因是由若干个编码区和非编码区互相间隔开但又连续镶嵌而成的结构，去除非编码区再连接后，为一个完整蛋白质编码外显子(exon)： 在断裂基因及其初级转录产物上出现，并可表达为成熟RNA的核酸序列内含子(intron)： 隔断基因线性表达而在剪接过程中被除去的核酸序列split gene: most structural genes of eukaryotes are compose

23、d of coding regions (exons) which are interrupted by intervening sequences (introns). In mRNA processing, the flanking exons are joined while the introns are removed.Exon: sequences that are present in the primary RNA transcript and in the mature RNA molecule are calledIntron: internal sequences tha

24、t are removed from the primary RNA transcript are called4. mRNA editing 基因转录产生的mRNA分子中，由于核苷酸的缺失，插入或置换，基因转录物的序列不与基因编码序列互补，使翻译生成的蛋白质的氨基酸组成，不同于基因序列中的编码信息，这种现象称为RNA编辑。 RNA编辑使得一个基因序列有可能产生几种不同的蛋白质，这可能是生物在长期进化过程中形成的、更经济有效地扩展原有遗传信息的机制。Post-transcriptional modification of tRNAtRNA的转录后修饰过程：1、剪接：在RNase P作用下，切除

25、5序列2、甲基化：A-mA3、还原反应：U-D4、核苷内的转位反应：U-5、脱氨反应：A-IPost-transcriptional modification of rRNA rRNA基因（rDNA）属于丰富基因族的序列，即染色体上存在的一些相似或完全一样的串联基因单位的重复，5srRNA和45srRNA分别由两种RNA pol合成 rDNA的转录和转录后修饰都在核仁中完成，转录修饰后的各种rRNA分子与核蛋白体蛋白组成核蛋白体而转运到胞质用于蛋白质的合成核酶Ribozyme (catalytic RNA) is a category of RNA molecules which have s

26、elf-splice catalytic function 具有自身催化作用的小RNA分子 In 1978 Altman was studying an RNA-cutting enzyme from the bacterium Escherichia coli. This enzyme, named RNAse P by biochemists, is composed of a complex between one protein and one RNA molecule. When Altman and his co-workers chemically split RNAse P a

27、nd separated the protein from the nucleic acid, the enzyme was no longer functional. However, he could restore the enzymatic activity by remixing the two different components. This was the first time that an RNA molecule had been shown to be necessary for a catalytic reaction. Cech was studying the

28、splicing of RNA in a unicellular organism called Tetrahymena thermophila. He discovered, much to his surprise, that when he put an unprocessed RNA molecule into a test tube in the absence of protein, it started to splice itself. In other words, the RNA molecule could cut itself into pieces and Join

29、the genetically important RNA fragments together again. Through the discovery of this chemically very complicated self-splicing reaction, Cech in 1982 became the first to show that RNA molecules can have a catalytic function. The discovery of catalytic RNA has altered the central dogma of the biosci

30、ences. Moreover, it has already had a profound influence on our understanding of how life on earth began and developed. It is very likely that the RNA molecules were the first biomolecules to contain both the genetic information and play a role as biocatalysts. Catalytic RNA will probably provide ge

31、ne technology with a new tool. In particular, it is hoped that gene shears, by cutting and destroying virus RNA, will protect organisms against viral infections. 小RNA干扰现象研究基础： 小RNA可以通过降解与之互补的靶mRNA序列而沉默其表达 This years Nobel Prize awards discovery and development of the green fluorescent protein, GFP.T

32、he Nobel Prize in Chemistry 2008 Osamu Shimomura, Japanese citizen. Born 1928 in Kyoto, Japan. Ph.D. in organic chemistry 1960 from Nagoya University, Japan. Professor emeritus at Marine Biological Laboratory (MBL), Woods Hole, MA, USA and Boston University Medical School, MA, USA. Osamu Shimomura first isolated GFP from the jellyfish Aequorea victoria, which drifts with the currents off the west coast of North Americ