Molecular Biology 分子生物学.ppt

Molecular Biology in Medicine医学分子生物学,许正平zpxuzju.edu.cn,http:/www.nobelprize.org/nobel_prizes/chemistry/laureates/2012/popular-chemistryprize2012.pdf,http:/www.nobelprize.org/nobel_prizes/chemistry/laureates/2012/popular-chemistryprize2012.pdf,一、导论,定义：从分子水平研究人体在正常和疾病状态下生命活动及其规律的一门科学重点：人体生物大分子和大分子体系的结构、功能、相互作用及其同疾病发生、发展的关系,医学分子生物学,分子医学 Molecular Medicine,The greatest intellectual revolution of the last 40 years may have taken place in biology. Can anyone be considered educated today who does not understand a little about molecular biology? F. H. Westheimer (Harvard University),Molecular medicine is a broad field, where physical, chemical, biological and medical techniques are used to describe molecular structures and mechanisms, identify fundamental molecular and genetic errors of disease, and to develop molecular interventions to correct them.,Molecular Medicine,GFP and other FPs (fluorescent proteins),Telomere DNA protects the chromosomes,Vesicle trafficing: get the right molecules to the right place at the right time,http:/www.nobelprize.org/nobel_prizes/medicine/laureates/2013/press.html,Gene & Genome基因与基因组 Gene Transcription 基因转录 RNA Splicing & Editing RNA剪切与加工 Protein Synthesis & Processing 蛋白质合成与加工 Protein Structure & Function蛋白质结构与功能 non-coding RNA非编码RNA,分子生物学主要研究内容,分子生物学主要研究技术,分离、纯化（主要是生物大分子） 克隆、表达 序列分析 （Sanger Seq Western blotting 微阵列技术：genechip, microarray, protein chip,分子生物学主要研究技术,基因操纵技术：Gene knock-out/knock-in RNA interference (RNAi)蛋白质翻译调控：ribosome profiling; GTI-seq蛋白质鉴定：质谱蛋白质相互作用：酵母双杂交、免疫共沉淀（Co-IP）、 pull-down、FRET、表面等离子共振技术(SPR) 蛋白质与核酸相互作用：ChIP、ChIP-on-chip 研究生物大分子三维结构常用的实验手段： X射线晶体学、核磁共振、电子显微学、原子力显微镜 以及X射线小角散射等。,二、基本知识介绍,人类基因与基因组,人类基因典型结构,中心法则 The Central Dogma,人细胞中的基因表达,FACT 1: an uniform genome in almost every cell of human body,FACT 3: the shape and function of each type of cell are different,FACT 2: the proteome in each type of cell is different,Some Facts in Human Cells,the types and amounts of the various proteins in a cell,the concentration of mRNA and the frequency at which the mRNA is translated,which genes are transcribed and their rateof transcription in a particular cell type,TRUTH: the gene is differentially expressed,regulation,same genome in all cells of an organism,regulation,regulation,Gene differential expression,Gene Expression Occurs by a Two-Stage Process,Transcription: generates a single-stranded RNA identical in sequence with one of the strands of the duplex DNA Three principal classes of products:message RNA (mRNA)transfer RNA (tRNA)ribosomal RNA (rRNA) Principle: complementary base pairingTranslation: converts the nucleotide sequence of an RNA into the sequence of amino acids comprising a protein each mRNA contains at least one coding region that is related to a protein sequence,DNA (gene)RNA polymeraseRegulatory Proteins,Key Players,promoter,A,startpoint,terminator,Transcription Unit,template,upstream,downstream,enhancer,Gene Transcription,Primary transcript is the original unmodified RNA product correspondingto a transcription unit.Promoter is a region of DNA involved in binding of RNA polymerase to initiate transcription.RNA polymerases are enzymes that synthesize RNA using a DNA template(formally described as DNA-dependent RNA polymerases).Terminator is a sequence of DNA, represented at the end of the transcript,that causes RNA polymerase to terminate transcription.Transcription unit is the distance between sites of initiation and termination by RNA polymerase; may include more than one gene.,Key Terms,Gene Transcription,Transcription in eukaryotic cells is divided into three classes. Each class is transcribed by a different RNA polymerase: RNA polymerase I: RNA polymerase II: RNA polymerase III:,RNA Polymerase,Transcription in eukaryotic cells is divided into three classes. Each class is transcribed by a different RNA polymerase: RNA polymerase I: rRNA; resides in the nucleolus RNA polymerase II: mRNA, snRNA; locates in the nucleoplasm RNA polymerase III: tRNA and other small RNAs; nucleoplasm,RNA Polymerase,The promoters for RNA polymerases I and II are (mostly) upstreamof the startpoint, but some promoters for RNA polymerase III lie downstream of the startpoint. Each promoter contains characteristic sets of short conserved sequences that are recognized by the appropriate class of factors. RNA polymerases I and III each recognize a relatively restricted setof promoters, and rely upon a small number of accessory factors. Promoters utilized by RNA polymerase II show more variation in sequence, and are modular in design.,Promoter,Short sequence elements (cis-acting elements): bind by accessory factors (transcription factors)The regulatory region might exist in the promoters of certain eukaryotic genes.Location: usually upstream and in the vicinity of the startpoint.These sites usually are spread out over a region of >200 bp. common: used constitutivelyspecific: usage is regulated; define a particular class of genesThese sites are organized in different combinations,Cis-acting Element,Enhancer element is a cis-acting sequence that increases the utilization of (some) eukaryotic promoters.The components of an enhancer resemble those of the promoter. Involve in initiation, but far from startpoint. Are targets for tissue-specific or temporal regulation.Function in either orientation and in any location (upstream or downstream) relative to the promoter.,Enhancer,two characteristics:1. the position of the enhancer need not be fixed.2. it can function in either orientation.,The Difference between Promoter and Enhancer,The distinction between promoters and enhancers is operational, rather than imply a fundamental difference in mechanism,Most Eukaryotic Genes Are Regulated by Multiple Transcription-Control Elements,(a) Genes of multicellular organisms contain both promoter-proximal elements and enhancersas well as a TATA box or other promoter element. Enhancers may be either upstream or downstream and as far away as 50 kb from the transcription start site. In some cases, promoter-proximal elements occur downstream from the start site as well. (b) Most yeast genes contain only one regulatory region, called an upstream activating sequence (UAS), and a TATA box, which is 90 base pairs upstream from the start site.,Fact: Regulatory elements in eukaryotic DNA often are many kilobases from start sites,Finding Regulatory Element in Eukaryotic DNA,Transcription Factor,Any protein that is needed for the initiation of transcription, but which is not itself part of RNA polymerase, is defined as a transcription factor. binds to DNA (trans-acting factor): recognize cis-acting elementsinteracts with other protein: recognize RNA pol, or another factor,The common mode of regulation of eukaryotic transcription is positive: a transcription factor is provided under tissue-specific control to activate a promoter or set of promoters that contain a common target sequence. Regulation by specific repression of a target promoter is less common.,Accessory factors are needed for initiation, principally responsible for recognizing the promoter. Interaction with DNA, RNA polymerase, and/or another factors.Three groups:General factorsUpstream factorsInducible factors,Another name: accessory factor,general factors: required for the mechanics of initiating RNA synthesis at all promoters; form a complex surrounding the startpoint with RNA pol, and determine the site of initiation. basal transcription apparatus (pol + GF) upstream factors: DNA-binding proteins that recognize specific short consensus elements located upstream of the startpoint. not regulated; ubiquitous; act upon any promoter that contains the appropriate binding site on DNA. inducible factors: function in the same general way as the upstream factors. have a regulatory role: control transcription patterns in time and space,Accessory Factors,Four Stages in Transcription,