二、基本知识介绍

1. 二、基本知识介绍

2. Hierarchies of Genome Organization

3. 人类基因与基因组

4. 人类基因与基因组

5. 中心法则 The Central Dogma

6. 人细胞中的基因表达

7. 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

8. 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

9. 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 Main products: message RNA (mRNA) transfer RNA (tRNA) ribosomal RNA (rRNA) non-coding RNA (ncRNA) 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

10. （一）基因转录及其调控

11. The three stages of DNA transcription

12. Gene Structure and its Transcription

13. http://en.wikipedia.org/wiki/Transcription_(genetics)

14. I. Transcription Key Players DNA (gene) RNA polymerase Regulatory Proteins (transcriptional factors) Non-coding RNAs enhancer promoter terminator startpoint template A Transcription Unit upstream downstream

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

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

24. I. 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. I. 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. I. 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. Regulation of transcription Regulation Levels On the genome Which gene(s) to be transcribed? Basic events: Protein binding and/or modification Epigenetics 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!

28. II. 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

29. II. Regulation of transcription Epigenetics Arthur Riggs: the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence NIH: refers to both heritable changes in gene activity and expression (in the progeny of cells or of individuals) and also stable, long-term alterations in the transcriptional potential of a cell that are not necessarily heritable. Cold Spring Harbor meeting: stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence

30. II. Regulation of transcription Epigenetics

31. II. Regulation of transcription Epigenetics: main players • DNA methylation • Histone modification: • methylation • acetylation • … • non-coding RNAs

32. II. 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?

33. II. 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

34. II. 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.

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

36. II. 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

37. II. 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

38. III. 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 • RNA editing: insertion, deletion, and base substitution of nucleotide

39. III. 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.

40. III. 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.

41. III. 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

42. III. 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?

43. III. 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

44. III. RNA Processing Trans-(intermolecular) splicing Splicing is usually cis-reaction (intramolecular), but trans- (intermolecular) splicing have been found (very rare). Trans-splicing is a special form of RNA processing in eukaryotes where exons from two different primary RNA transcripts are joined end to end and ligated. trypanosomes and euglenoids: all the mRNAs Caenorhabditis elegans: 10-15% of the mRNAs Human?

45. （二）蛋白质翻译及加工

46. IV. 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

47. 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

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

49. 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

50. V. Initiation of Protein Synthesis New Progress • Initiation does not necessarily start from AUG Science. 2012, 336(6089):1719-23.