Molecular Biology in Medicine 医学分子生物学

1. Molecular Biology in Medicine医学分子生物学 许正平 zpxu@zju.edu.cn

2. http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2012/popular-chemistryprize2012.pdfhttp://www.nobelprize.org/nobel_prizes/chemistry/laureates/2012/popular-chemistryprize2012.pdf

3. http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2012/http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2012/ popular-chemistryprize2012.pdf

4. 一、导论

5. 医学分子生物学 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 分子医学 Molecular Medicine

6. 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)

7. Molecular Medicine 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.

13. GFP and other FPs (fluorescent proteins)

16. Telomere DNA protects the chromosomes

23. 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

28. 分子生物学主要研究内容 • 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

29. 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 & Next Generation Seq) • PCR（多聚酶链式反应 ） • 凝胶电泳：琼脂糖凝胶电泳；SDS－聚丙烯酰胺凝胶电泳 （SDS-PAGE）；等电聚焦电泳；双向电泳 • 印迹技术：Southern blotting; Northern blotting; Western blotting • 微阵列技术：genechip, microarray, protein chip

30. 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。

31. 二、基本知识介绍

32. 人类基因与基因组

33. 人类基因典型结构

34. 中心法则 The Central Dogma

35. 人细胞中的基因表达

36. Some Facts in Human Cells FACT 1: an uniform genome in almost every cell of human body FACT 2: the proteome in each type of cell is different FACT 3: the shape and function of each type of cell are different

37. TRUTH: the gene is differentially expressed same genome in all cells of an organism regulation which genes are transcribed and their rate of transcription in a particular cell type regulation the concentration of mRNA and the frequency at which the mRNA is translated regulation the types and amounts of the various proteins in a cell Gene differential expression

38. 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 pairing • Translation: 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

39. Gene Transcription Key Players DNA (gene) RNA polymerase Regulatory Proteins enhancer promoter terminator startpoint template A Transcription Unit upstream downstream

40. Gene Transcription Key Terms Primary transcript is the original unmodified RNA product corresponding to 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.

41. RNA Polymerase • 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:

42. 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

43. Promoter The promoters for RNA polymerases I and II are (mostly) upstream of 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 set of 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.

44. Cis-acting Element 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 constitutively specific: usage is regulated; define a particular class of genes These sites are organized in different combinations

45. Enhancer • 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. • two characteristics: • 1. the position of the enhancer need not be fixed. • 2. it can function in either orientation.

46. The Difference between Promoter and Enhancer The distinction between promoters and enhancers is operational, rather than imply a fundamental difference in mechanism

47. Most Eukaryotic Genes Are Regulated by Multiple Transcription-Control Elements (a) Genes of multicellular organisms contain both promoter-proximal elements and enhancers as 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.

48. Finding Regulatory Element in Eukaryotic DNA Fact: Regulatory elements in eukaryotic DNA often are many kilobases from start sites

49. 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 elements interacts 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.

50. Another name: accessory factor • Accessory factors are needed for initiation, principally • responsible for recognizing the promoter. • Interaction with DNA, RNA polymerase, and/or another • factors. • Three groups: • General factors • Upstream factors • Inducible factors