分子生物学Molecular+Biology-课件

1、课程安排章节课程内容原章节1绪论12基因组与基因组学93重组与转座74原核生物的转录及调控35真核生物的转录及调控46转录后加工及基因表达调控57蛋白质翻译6自学分子生物学研究方法2后半部分课程安排专题课程内容1真核生物的RNA聚合酶及其启动子2真核生物的转录因子、增强子3染色质结构及其对基因转录的影响4mRNA的加工：剪接、加帽与加尾5其它RNA的加工、转录后基调控6蛋白质翻译-分子生物学研究方法认识分子生物学的重要地位提纲挈领，把握重点理解胜于记忆重视基础研究方法尊重历史人物泛读寄语What is Molecular Biology ?狭义的分子生物学就是基因的分子生物学Replicati

2、onSame Genome, Differentiated Expression分子生物学的核心是基因的表达与调控真核生物与原核生物基因表达的异同真核生物转录与翻译隔开；原核生物基因是多顺反子；真核生物转录后加工。See the movie. 真核生物的基因转录及其调控转录过程的本身在真核生物与原核生物中是相同的，但所用的转录“机器”存在差别（RNA聚合酶、启动子、转录因子）；真核生物转录机器的复杂性为真核基因的调控提供更多的控制点。. 真核生物的基因转录及其调控一、 真核生物的RNA聚合酶 Question1： 真核生物的RNA聚合酶(polymerase)与原核生物一样吗？Clues: 真

3、核生物核糖体基因具有如下特点： GC含量高（60%），其它基因DNA GC含量40%； 拷贝极多（几百-两万）； 定位在核仁Surmise: 真核生物中可能不止一种RNA聚合酶分离真核细胞的RNA聚合酶Robert Roeder & William Rutter1942-University of Washington1928-University of Washington2003 Lasker Award2000 Lasker Award (scientists in Chiron corporation)William Rutter1969-1991 UCSFChiron corpora

4、tion founderRobert Weaverpost-doc 1969-1971UCSF William Rutter的传奇一生Between 1952 and 1968, Rutter held positions at the University of Wisconsin, the Karolinska Institutet, University of Illinois, Stanford University and University of Washington. In 1969, UCSF succeeded in recruiting Rutter to UCSF to

5、 develop a new department of Biochemistry and Biophysics. He completed structural and functional analysis and cloning of the rat and human insulin.In 1981 Rutter and two colleagues founded Chiron.He conducted Hepatitis B research, which resulted in development with Chiron and Merck of the vaccine fo

6、r Hepatitis B, the first recombinant vaccine. Also at Chiron, he worked on the first sequencing of the HIV genome in 1984, and discovered, sequenced, and cloned the Hepatitis C virus in 1987. This opened the way for the development of diagnostic tests, therapeutic drugs, and vaccines against these v

7、iruses.After his retirement from UCSF in 1991, William Rutter began a second distinguished career as spokesman and developer of the biotechnology industry, and subsequent years have brought new work and more recognition. 分离真核细胞的RNA聚合酶Nature 1969; 224:234-237材料：海胆胚和大鼠肝细胞核提取物海胆，徐辉 2011-10-22摄于外伶仃岛离子交换

8、层析原理示意图注：不同颜色代表电荷不同的蛋白质方法：离子交换层析阴离子交换树脂分离蛋白质的原理将阴离子交换树脂（带正电荷）颗粒填充在层析管内，可吸附带负电荷的蛋白质，由于各种蛋白质所带电荷的种类和数量不同，它们被吸附的程度也不同。用含阴离子（如Cl-)的溶液洗柱，含电荷少的蛋白质首先被洗脱下来，增加阴离子浓度，含电荷多的蛋白质也被洗脱下来。不断增加离子浓度，收集各种组分，对每种收集组分进行分析。详细可查阅：5.1 离子交换层析Nature 1969; 224:234-237海胆胚提取物层析分析结果问题：为什么用UMP?大鼠肝提取物层析分析结果Nature 1969; 224:234-237大鼠

9、肝提取物核质和核仁馏分层析分析结果PNAS 1970; 65:675-682大鼠肝细胞核质馏分大鼠肝细胞核仁馏分Harvard UniversityRichard Burgess & Andrew Travers 分离原核细胞的RNA聚合酶Now: University of WisconsinNow: University of Cambridge Harvard UniversityNature 1969(Jan); 221: 43 - 46 Nature 1969(Jan); 221: 43 - 46 中文第四版书上标注有误Robert RoederRichard Burgess Ph.

10、D., 1969UCSFPh.D., 1969, Harvard UniversityAndrew Travers post-doc 1969 Harvard University年轻有为、硕果累累的科学家科研之巅峰：一年四篇natureQuestion2： 真核生物的三种RNA聚合酶各转录什么RNA?Strategies: 纯化三种RNA聚合酶，分析它们体外可转录哪些特定的基因;抑制各种RNA聚合酶的活性，检测各种RNA基因的转录变化。巧妙！-鹅膏蕈（ xn ）碱（amanitin ）致死性毒蘑菇鬼笔鹅膏菌（Amanita phalloides）产生的具有双环结构的八肽毒素此菌极毒，中毒后潜

11、伏期长。发病初期以胃肠道病症。恶心、呕吐、腹痛、腹泻、此后一、两天似乎病愈，实际上毒素进一步损害肝、肾、心脏、肺、大脑中枢神经系统。病情很快恶化，出现呼吸困难，烦燥不安，谵语、面肌抽搐、小腿肌肉痉挛。病情进一步加重，出现肝、肾细胞损害，黄胆，急性肝炎，肝肿大及肝萎缩，最后昏迷。死亡率高达50%以上，甚至100%。地理分布生长在林地，特别是橡树林或山毛榉树林里，夏秋季在阔叶林中地上也有单生或群生。分布在我国江苏 江西 湖北 安徽 福建 湖南 广东 广西 四川 贵州 云南等地。 致命白毒伞（毒蕈类）外形与一些传统的食用蘑菇较为相似，极易引起误食，喜欢在黧蒴树的树荫下群生。黧蒴树在广州地区白云山、天

12、麓湖、华南植物园等山地均有分布。在新鲜毒菇中毒素含量很高，50克左右的白毒伞菌体所含毒素便足以毒死一个成年人。中毒者死亡率高达90以上，是历年广州地区毒菇致死事件的罪魁祸首。Decreased RNA content in mouse liver nuclei after intoxication with -amanitin.L. Fiume, F. StirpeIstituto di Patologia generale, Universit di Bologna Bologna, ItalyBiochimica et Biophysica Acta (BBA) Sep 1966Effec

13、t of alpha-amanitin on ribonucleic acid synthesis and on ribonucleic acid polymerase in mouse liver. F. Stirpe, L. FiumeThe Biochemical journal Jun1967Biological Sciences: Specific Action of -Amanitin on Mammalian RNA Polymerase ProteinJacob, S. T. , Sajdel, E. M. , and Munro, H. N Department of Nut

14、rition and Food Science, Massachusetts Institute of Technology.Nature Jan 1970Specific Inhibition of Nuclear RNA Polymerase II by -AmanitinThomas J. Lindell, Fanyela Weinberg, Paul W. Morris, Robert G. Roeder and William J. RutterDepartment of Biochemistry and Biophysics, University of California, S

15、an Francisco.Science Oct 1970Role of DNA-dependent RNA polymerase 3 in the transcription of the tRNA and 5S RNA genes.William J. Rutter and Robert G. Roeder Department of Biochemistry and Biophysics, University of California, San Francisco.PNASMay 1974不同浓度的-Amanitin对三种RNA聚合酶活性的影响PNAS 1974; 71(5): 17

16、9094.PNAS 1974; 71(5): 179094.-Amanitin对小RNA合成的影响5S= 5SrRNA, 4S=4StRNA.microRNA75%-85%15%3-5%Question3： 真核生物的三种RNA聚合酶的组成是什么?Strategies: 层析分离纯化各种RNA聚合酶，SDS变性胶电泳，观测各蛋白亚基的大小。Hager, G L; Holland, M J; Rutter, W JHigh yields of each enzyme activity are obtained, allowing the preparation of approximately

17、10 mg of polymerase I, 25 mg of polymerase II, and 12 mg of polymerase III from 1.2 kg of cells (wet weight). Isolation of ribonucleic acid polymerases I, II, and III from Saccharomyces cerevisiae. Biochemistry1977; 16(1): 1-8酵母三种RNA聚合酶的电泳图Biochemistry1977; 16(1): 1-8IIIIIIQuestion4： 层析法分离的蛋白产物中是否包含

18、各种RNA聚合酶的所有亚基（或组分）?Strategies: 免疫沉淀法分离RNA聚合酶复合物，SDS变性胶电泳检测各亚基。表位标签法（epitope tagging）： 通过DNA重组，将外源抗原决定簇基因片段（tag）与RNA聚合酶的一个亚基的基因片段融合，导入该亚基缺失的细胞株中，通过抗体识别该融合蛋白(tag片段)，将蛋白复合物免疫沉淀下来。WTTaggedTagSDS变性胶电泳32P标记可检测是不是磷酸化的蛋白酵母RNA聚合酶的亚基结构Kolodziej et al., Mol. Cell Biol. 10 (May 1990) p. 1917酵母RNA聚合酶的亚基结构Sayre e

19、t al., J. Biol. Chem. 267 (15 Nov 1992) p. 23379RNA聚合酶大亚基的异质性（1）蛋白质纯化过程中C末端结构域丢失（2）蛋白质磷酸化引起电泳速度改变Question5：三种RNA聚合酶各亚基的功能是什么?Strategies: 纯化推定的各种RNA聚合酶的亚基，重组成有活性的酶，从而判断各亚基是否必需;X亚基太多Strategies: 序列比对，通过同源性推断各亚基的功能；Regions of homology between yeast Rpb1 and the E. coli polymerase subunit and between Rpb

20、2 and the E. colipolymerase subunit. The regions of homology are shown as red boxes; the carboxyl terminal domain (CTD) in Rpb1 is in green.Rpb1Rpb2The subunits of RNA polymerases ProkaryoticBacteriaArcheaEukaryoticRNAP1RNAPRNAPA/ABDLK+6 othersRPA1RPA2RPC5RPC9RPB6RPB1RPB2RPB3RPB11RPB6RPC1RPC2RPC5RPC

21、9RPB9+9 others+7 others+11 othersStrategies: 序列比对，通过同源性推断各亚基的功能；克隆各种RNA聚合酶的亚基， 逐个突变，判断哪些亚基是聚合酶酶活性的必需组分。共有亚基核心亚基非必需亚基真核生物RNA聚合酶各亚基发挥的作用Question6：真核RNA聚合酶的三维结构是什么样子?Roger D. Korberg，中山大学诺贝尔大师系列讲坛，2011-11-17All figures from10-10 to 10-18 in Molecular Biology (Weaver, 4e) are refered to Roger D. Korberg

22、, the 2006 laureate of the Nobel Prize in ChemistryPatrick Cramer, David A. Bushnell, Roger D. Kornberg. Structural Basis of Transcription: RNA Polymerase II at 2.8 ngstrom Resolution. Science 2001;292: 1863-1876.Averell L. Gnatt, Patrick Cramer, Jianhua Fu, David A. Bushnell, Roger D. Kornberg. Str

23、uctural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Resolution. Science 2001;292: 1876-1882.He created three-dimensional images of RNA Polymerase II using X-ray crystallographyRoger Korberg and his father, Arthur Korberg (1918-2007), the 1959 laureate of the Nobel Prize in

24、 Physiology or Medicine.Nobel Prize is not hereditary On November 21, 1943, Kornberg married Sylvy Ruth Levy, also a biochemist of note. She worked closely with Kornberg and contributed significantly to the discovery of DNA polymerase. The day after Arthur Korberg was awarded the Nobel prize, she wa

25、s quoted in a newspaper as saying I was robbed.The Family of ScientistsArthur and Sylvy Kornberg had three sons: Roger David Kornberg (1947), Thomas B. Kornberg (1948), and Kenneth Andrew Kornberg (1950). Thomas discovered DNA polymerase II and III in 1970 and is now a professor at the University of

26、 California, San Francisco. Kenneth is an architect specializing in the design of biomedical and biotechnology laboratories and buildings.The Family of ScientistsLate in 1957, Kornbergs manuscripts describing the test-tube synthesis of DNA from precursor molecules by an enzyme DNA polymerase were re

27、jected by The Journal of Biological Chemistry. Reviewers thought it premature to call the product DNA. In the spring of 1958, however, a new editor stepped in and accepted the papers. Less than two years later, Kornbergs discovery, were recognized by the Nobel Prize in Physiology or Medicine.获得诺奖最快的

28、生物学家 Nature 450, 809 (6 December 2007) Nature 450, 809 (6 December 2007) 谁真谁假？ Nature 450, 1176 (20 December 2007) 获得诺奖最快的科学家是谁？ 获得诺奖最迟的科学家是谁？ 真核生物RNA聚合酶的三维结构特征T.aquatics(水生嗜热菌)S.cerevisiae原核生物与真核生物RNA聚合酶晶体结构对比 Question： 细胞中各种RNA的含量：rRNA约占80%，tRNA占15%，mRNA占3-5%，分别由RNA聚合酶、转录（5.8S rRNA除外）；但RNA聚合酶、的比例大

29、概是：10:12:25（yeast），为什么mRNA的比例那么少？ Possible Answers： 翻译过程是非常高效的，一条mRNA可翻译出很多蛋白质。 mRNA的产物是蛋白质，是细胞活动的执行者和决定者，每时每刻细胞内蛋白质的种类和数量都是精确调控的。因此，mRNA转录的开启必需受到严格的控制。. 真核生物的基因转录及其调控二、真核生物基因的启动子转录必需因素： DNA模板 RNA聚合酶 启动子 转录因子转录启动所依赖的DNA序列 真核RNA聚合酶识别的启动子 I 类启动子RNA聚合酶I识别 II 类启动子RNA聚合酶II识别 III类启动子RNA聚合酶III识别5类元件，后4类是核心

30、启动子元件上游元件（upstream element） TFB识别元件（BRE） TATA box起始子（initiator）下游元件（downstream element） II类启动子（蛋白质等基因的启动子）+1-33 II类启动子 与原核生物的启动子最为相似Up element- 33-10 box (TATA)-35 box (TTGACa)-10因子识别位点TBP识别TFB识别+1TATA box位于转录起始位点上游约25-30 bp（在酵母中可达30 300 bp），共同序列为TATAAA；功能：确定转录起始位点（TATA区下游25-30 bp左右）；有时甚至是决定整个启动子是否有活性。Effects of deletions in the SV40 early promoter箭头代表缺失的区域TATA box缺失后导致转录可在多个位点起始。S1核酸酶作图法：参见5.4章节缺失扫描是启动子定位的最常用研究方法Zhou et al, Oncogene 2011定点突变是鉴定转录因子结合位点的最常用方法Xu et al, Hepatology 2010TATA box有些基因的TATAbox序列中会出现G和C，如beta-球蛋白基因(CATA).有两类基因甚至缺