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. 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)

3. 分子生物学主要内容 • Genetic Information Transfer 遗传信息的传递 • 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 • Regulation of Gene Expression 基因表达的调控(包括miRNA、RNAi)

4. 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 • 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 • 基因操纵技术：Gene knock-out/knock-in RNA interference (RNAi)

5. 分子生物学主要研究技术 • 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) • 蛋白质鉴定：质谱 • 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。

6. 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

7. 教材：医学分子生物学（第3版） 参考书：Molecular Cell Biology Gene More Information: Literature Internet

8. I. Introduction The Central Dogma

9. I. Introduction How many genes in the human genome? Gene Expression

10. I. Introduction FACT 1: an uniform genome in almost every cell of an organism FACT 2: the proteome in each type of cell is different FACT 3: the shape and function of each type of cell are different

11. I. Introduction FACT 1: an uniform genome in almost every cell of an organism FACT 2: the proteome in each type of cell is different FACT 3: the shape and function of each type of cell are different the actions and properties of each cell type are determined by the proteins it contains transcription of different genes largely determines the actions and properties of cells

12. I. Introduction 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

13. I. Introduction 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

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

15. II. 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.

16. II. Transcription 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:

17. II. Transcription 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

18. II. Transcription 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.

19. II. Transcription 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

20. II. Transcription 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.

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

22. II. Transcription 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.

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

24. II. Transcription 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.

25. II. Transcription 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

26. II. Transcription Accessory Factors • 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

27. II. Transcription Four Stages in Transcription

28. III. Regulation of transcription Regulation Levels On the genome Which gene(s) to be transcribed? Basic events: Protein binding and/or modification 2. On a specific gene If the gene can be transcribed successfully? 3. On a transcript If the transcript could be correctly spliced? If the transcript could be correctly edited? Key determinant: Cell Signaling!

29. III. Regulation of transcription Potential regulation points 5 potential control points: Activation of gene structure ↓ Initiation of transcription ↓ Processing the transcript ↓ Termination of transcription ↓ Transport to cytoplasm “Active” Structure Major Control Point Alternative Splicing the overwhelming majority of regulatory events occur at the initiation of transcription

30. III. Regulation of transcription Regulatory Proteins the overwhelming majority of regulatory events occur at the initiation of transcription Key player: regulatory transcription factors • Two questions: • How does the transcription factor identify its group of target genes? • How is the activity of the transcription factor itself regulated in • response to intrinsic or extrinsic signals?

31. III. Regulation of transcription Answer to question 1 The genes share common response element Structure feature: contain short consensus sequence Examples: HSE: heat shock response element; recognized by HSTF GRE: glucocorticoid response element SRE: serum response element MRE: metal response element

32. III. Regulation of transcription Regulatory region in MT gene ? = MTF-1 BLE: basal level element; TRE: TPA response element General Principle: any one of several different elements, located in either an enhancer or promoter, can independently activate the gene.

33. III. Regulation of transcription Answer to question 2 Signal transduction • Key events: • Protein synthesis • Protein modification • Ligand binding • Protein cleavage • Inhibitor release • Mutation

34. III. Regulation of transcription Regulation Modes of Transcription Factor The activity of a regulatory transcription factor may be controlled by synthesis of protein, covalent modification of protein, ligand binding, or binding of inhibitors that sequester the protein or affect its ability to bind to DNA. mutations of the transcription factors give rise to factors that inappropriately activate, or prevent activation, of transcription

35. III. Regulation of transcription Eukaryotic transcriptional control operates at three levels during the stage of initiation 1. changes in chromatin structure directed by activators and repressors 2. modulation of the levels of activators and repressors (gene expression) 3. change the activities of activators and repressors Gene differential expression

36. IV. RNA Processing INTRODUCTION • Facts: • Genes are interrupted, and mRNAs are uninterrupted • The primary transcript has the same organization as the gene • Most mRNAs have 5’ cap and 3’ poly(A) tail • Heterogeneous nuclear RNAs (hnRNA) exist in the nucleus • RNA contains rare bases • Mechanism: • RNA splicing: remove intron • RNA modification: 5’ capping, 3’ polyadenylation, base modification

37. IV. RNA Processing INTRODUCTION • The initial primary transcript synthesized by RNA polymerase II • undergoes several processing steps before a functional mRNA • is produced: • 5’ capping • 3’ cleavage/polyadenylation • RNA splicing RNA splicing is the process of excising the sequences in RNA that correspond to introns, so that the sequences corresponding to exons are connected into a continuous mRNA.

38. IV. RNA Processing Overview of mRNA Processing in Eukaryotes The poly(A) tail: ~250 A in mammals, ~150 in insects, ~100 in yeasts. For short primary transcripts with few introns, polyadenylation, cleavage, and splicing usually follows termination. For large genes with multiple introns, introns often are spliced out of the nascent RNA before transcription of the gene is complete.

39. IV. RNA Processing The splicing snRNPs associate with the pre-mRNA and with each other in an ordered sequence to form spliceosome The spliceosomal splicing cycle ATP is needed to provide the energy necessary for rearrangements of the spliceosome structure

40. IV. RNA Processing Alternative splicing Definition: a single gene gives rise to more than one mRNA sequence • Mechanisms: • use of different startpoints or termination sequences • a single primary transcript is spliced in more than one way, and internal exons are substituted, added, or deleted Key: what controls the use of such alternative pathways? Proteins? ncRNA?

41. IV. RNA Processing Alternative splicing Definition: a single gene gives rise to more than one mRNA sequence • Mechanisms: • use of different startpoints or termination sequences • a single primary transcript is spliced in more than one way, and internal exons are substituted, added, or deleted Key: what controls the use of such alternative pathways? Protein(s)!

42. IV. RNA Processing The Troponin （肌钙蛋白） T (muscle protein) pre-mRNA is alternatively spliced to give rise to 64 different isoforms of the protein Constitutively spliced exons (exons 1-3, 9-15, and 18) Mutually exclusive exons (exons 16 and 17) Alternatively spliced exons (exons 4-8) Exons 4-8 are spliced in every possible way giving rise to 32 different possibilities Exons 16 and 17, which are mutually exclusive, double the possibilities; hence 64 isoforms

43. IV. RNA Processing Trans-(intermolecular) splicing Splicing is usually cis-reaction (intramolecular), but trans- (intermolecular) splicing have been found (very rare). These reactions probably occur by splicesome formation with the appropriate site sequences on each molecule. trypanosomes and euglenoids: all the mRNAs Caenorhabditis elegans: 10-15% of the mRNAs Human?

44. V. Initiation of Protein Synthesis Initiation of Protein Synthesis Critical event: begin protein synthesis at the start codon, thereby setting the stage for the correct in-frame translation of the entire mRNA. Main mechanisms: Base pairing between mRNA and rRNA Base pairing between mRNA and tRNA Met-tRNAiMet can only bind at the P site to begin synthesis Participants: Met-tRNAiMet mRNA IFs small subunit large subunit Protein translation

45. V. Initiation of Protein Synthesis Two types of methionine tRNA are found in all cells same aminoacyl-tRNA synthetase (MetRS) charges both tRNAs with methionine

46. V. Initiation of Protein Synthesis Eukaryotic initiation of protein synthesis

47. V. Initiation of Protein Synthesis PABI and eIF4 (4G and 4E) can interact on mRNA to circularize the molecule Model of protein synthesis on circular polysomes and recycling of ribosomal subunits

48. VI. Protein Processing Protein Maturation The nascent polypeptide chain must undergo folding and, in many cases, chemical modification and cleavage to generate the final protein Folding: Theoretically: any polypeptide chain containing n residues could, in principle, fold into 8n conformations. Fact: adopt a single conformation (native state) a single, energetically favorable conformation Mechanism: the amino acid sequence provides the information for protein folding Modification: N terminal C terminal Certain sites btw N and C terminus

49. VI. Protein Processing Protein Alteration Nearly every protein in a cell is chemically altered after its synthesis in a ribosome, thus alter its activity, life span, or cellular location of proteins, depending on the nature of the alteration. Two categories: chemical modification involves the linkage of a chemical group to the terminal amino or carboxyl groups or to reactive groups in the side chains of internal residues may be reversible Processing involves the removal of peptide segments and generally is irreversible

50. VI. Protein Processing Protein Modification The internal residues in proteins can be modified by attachment of a variety of chemical groups to their side chains: phosphorylation (Ser, Thr, Tyr) glycosylation (Asp, Ser, Thr) ubiquitination others Examples of modified internal residues produced by hydroxylation, methylation, and carboxylation