Welcome Each of You to My Molecular Biology Class

1. Welcome Each of You to My Molecular Biology Class

2. Molecular Biology of the Gene, 5/E--- Watson et al. (2004) Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods 2005-5-10

3. Part IV Regulation Ch 16: Regulation in prokaryotes Ch 17: Regulation in eukaryotes Ch 18: Regulation during development and in diseases (brief introduction) Ch 19: Comparative genomics and evolution of animal diversity(Not covered in the lecture)

4. Chapter 16 Regulation principles and How genes are regulated in bacteria Chapter 17 Basic mechanism of gene expression in eukaryotes Chapter 18 The mechanism of RNAi and the role of miRNA in development and cancergenesis

5. Surfing the contents of Part IV --The heart of the frontier biological disciplines

6. Chapter 17 Gene Regulation in Eukaryotes • Molecular Biology Course

7. 1. Gene Expression is Controlled by Regulatory Proteins (调控蛋白) Principles of Transcription Regulation Gene expression is very often controlled by Extracellular Signals,which are communicated to genes by regulatory proteins: • Positive regulators or activators INCREASE the transcription • Negative regulators or repressors DECREASE or ELIMINATE the transcription

8. Similarity of regulation between eukaryotes and prokaryote 1.Principles are the same: • signals (信号), • activators and repressors (激活蛋白和阻遏蛋白) • recruitment and allostery, cooperative binding (招募，异构和协同结合) 2. The gene expression steps subjected to regulation are similar, and the initiation of transcription is the most pervasively regulated step.

9. Difference in regulation between eukaryotes and prokaryote • Pre-mRNA splicing adds an important step for regulation. (mRNA前体的剪接) • The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart. (真核转录机器更复杂) • Nucleosomes and their modifiers influence access to genes. (核小体及其修饰体) • Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes. (真核基因有更多结合位点)

10. A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration Bacteria Yeast Human Fig. 17-1

11. Enhancer (激活元件) : a given site binds regulator responsible for activating the gene. Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals. Activation at a distance is much more common in eukaryotes. Insulators (绝缘子) or boundary elements (临界元件) are regulatory sequences between enhancers and promoters. They block activation of a linked promoter by activator bound at the enhancer, and therefore ensure activators work discriminately.

12. CHAPTER 17 Gene Regulation in eukaryotes 一、真核的转录激活蛋白的结构特征 The structure features of the eukaryotic transcription activators Topic 1: Conserved Mechanisms of Transcriptional Regulation from Yeast (酵母) to Mammals (哺乳动物)

13. The basic features of gene regulation are the same in all eukaryotes, because of the similarity in their transcription and nucleosome structure. • Yeast is the most amenable to both genetic and biochemical dissection, and produces much of knowledge of the action of the eukaryotic repressor and activator. • The typical eukaryotic activators works in a manner similar to the simplest bacterial case. • Repressors work in a variety of ways.

14. 1. Eukaryotic activators (真核激活蛋白) have separate DNA binding and activating functions【与原核相似】, which are very often on separate domains of the protein. Fig. 17-2 Gal4 bound to its site on DNA

15. Eukaryotic activators---Example 1: Gal4 • Gal4 is the most studied eukaryotic activator • Gal4 activates transcription of the galactose genes in the yeast S. cerevisae. • Gal4 binds to four sites (UASG) upstream of GAL1, and activates transcription 1,000-fold in the presence of galactose Fig. 17-3 The regulatory sequences of the Yeast GAL1 gene.

16. Eukaryotic activators---Example 1: Gal4 Experimental evidences showing that Gal4 contains separate DNA binding and activating domains. • Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription. • Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor, LexA activates the transcription of the reporter gene. Domain swap experiment 实验介绍系列1－Experiment introduction series

17. Domain swap experiment Moving domains among proteins, proving that domains can be dissected into separate parts of the proteins. Many similar experiments shows that DNA binding domains and activating regions are separable.

18. Box 1The two hybrid Assay (酵母双杂交) is used to identify proteins interacting with each other. （实验介绍系列2） Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4, respectively. Produce fusion proteins

19. 2. Eukaryotic regulators use a range of DNA binding domains, but DNA recognition involves the same principles as found in bacteria. • Homeodomain proteins • Zinc containing DNA-binding domain: zinc finger and zinc cluster • Leucine zipper motif • Helix-Loop-Helix proteins : basic zipper and HLH proteins

20. Bacterial regulatory proteins • Most use the helix-turn-helix motif to bind DNA target • Most bind as dimers to DNA sequence: each monomer inserts an a helix into the major groove. • Eukaryotic regulatory proteins • Recognize the DNA using the similar principles, with some variations in detail. • In addition to form homodimers (同源二聚体), some form heterodimers (异源二聚体) to recognize DNA, extending the range of DNA-binding specificity.

21. Homeodomain proteins: The homeodomain (同源结构域) is a class of helix-turn-helix DNA-binding domain and recognizes DNA in essentially the same way as those bacterial proteins. What is the same? Figure 17-5

22. Zinc containing DNA-binding domains (锌指结构域):Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4) Figure 17-6

23. Leucine Zipper Motif (亮氨酸拉链基序) : The Motif combines dimerization and DNA-binding surfaces within a single structural unit. Figure 17-7

24. Dimerization (二聚化)is mediated by hydrophobic interactions between the appropriately-spaced leucine (亮氨酸) to form a coiled coil structure

26. Helix-Loop-Helix motif: similar as in leucine zipper motif. Figure 17-8

27. myogenic factor:生肌调节蛋白是一种转录因子。

28. Because the region of the a-helix that binds DNA contains baisc amino acids residues, Leucine zipper and HLH proteins are often called basic zipper and basic HLH proteins. Both of these proteins use hydrophobic amino acid residues for dimerization.

29. 3. Activating regions(激活区域)are not well-defined structures • The activating regions are grouped on the basis of amino acids content. • Acidic activation region (酸性激活区域): contain both critical acidic amino acids and hydrophobic acids.yeast Gal4 • Glutamine-richregion (谷氨酰胺富集区): mammalian activator SP1 • Proline-rich region (脯氨酸富集区): mammalian activator CTF1

30. CHAPTER 17 Gene Regulation in eukaryotes 二、真核转录激活蛋白的招募调控方式和远距调控特征 Activation of the eukaryotic transcription by recruitment & Activation at a distance Topic 2: Recruitment of Protein Complexes to Genes by Eukaryotic Activators

31. Eukaryotic activators (真核激活蛋白) also work by recruiting (招募) as in bacteria, but recruit polymerase indirectly in two ways: 1. Interacting with parts of the transcription machinery. 2. Recruiting nucleosome modifiers that alter chromatin in the vicinity of a gene.

32. 1. Activators recruit the transcription machinery to the gene.

33. The eukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes, such as the Mediator and the TFⅡD complex. Activators interact with one or more of these complexes and recruit them to the gene. Figure 17-9

34. Box 2 Chromatin Immuno-precipitation (ChIP) (染色质免疫共沉淀)to visualize where a given protein (activator) is bound in the genome of a living cell.) （实验介绍系列3）

35. Activator Bypass Experiment (越过激活子实验)-Activation of transcription through direct tethering of mediator to DNA. （实验介绍系列4） Directly fuse the bacterial DNA-binding protein LexA protein to Gal11, a component of the mediator complex to activate GAL1 expression. Figure 17-10

36. At most genes, the transcription machinery is not prebound, and appear at the promoter only upon activation. Thus, no allosteric activation of the prebound polymerase has been evident in eukaryotic regulation。

37. 2. Activators also recruit modifiers that help the transcription machinery bind at the promoter • Modifiers direct recruitment of the transcriptional machinery • Modifiers help activate a gene inaccessibly packed within chromatin

38. Two types of Nucleosome modifiers : • Those add chemical groups to the tails of histones (在组蛋白尾上加化学基团), such as histone acetyl transferases (HATs) • Those remodel the nucleosomes (重塑核小体), such as the ATP-dependent activity of SWI/SNF. How do these modification help activate a gene ?

39. Two basic models for how these modification help activate a gene: • Remodeling and certain modification can uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome. • By adding acetyl groups, it creates specific binding sites on nucleosomes for proteins bearing so-called bromodomains (溴区结构域).

40. Fig 17-11 Local alterations in chromatin directed by activators （组蛋白乙酰化酶）

41. 溴区结构域蛋白 Figure 7-39 Effect of histone tail modification

42. Many enkaryotic activators－particularly in highereukaryotes－work from a distance. How? • Some proteins help, for example Chip protein in Drosophila. • The compacted chromosome structure help. DNA is wrapped in nucleosomes in eukaryotes.So sites separated by many base pairs may not be as far apart in the cell as thought. 3. Action at a distance: loops and insulators

43. Specific cis-acting elements called insulators (绝缘子) control the actions of activators, preventing the activating the non-specific genes

44. Insulators block activation by enhancers Figure 17-12

45. Transcriptional Silencing (转录沉默) Transcriptional Silencing is a specialized form of repression that can spread along chromatin, switching off multiple genes without the need for each to bear binding sites for specific repressor. Insulator elements (绝缘子元件) can block this spreading, so insulators protect genes from both indiscriminate activation and repression.[绝缘子的概念到此才介绍完毕] Application：A gene inserted at random into the mammalian genome is often “silenced”, and placing insulators upstream and downstream of that gene can protect the gene from silencing.

46. 4 Appropriate regulation of some groups of genes requires locus control region (LCR). Human and mouse globin genes are clustered in genome and differently expressed at different stages of development A group of regulatory elements collectively called the locus control region (LCR), is found 30-50 kb upstream of the cluster of globin genes. It binds regulatory proteins that cause the chromatin structure to “open up”, allowing access to the array of regulators that control expression of the individual genes in a defined order.

47. Figure 17-13 Please compare LCR with the Lac operon controlled gene expression in bacteria

48. Another group of mouse genes whose expression is regulated in a temporarily and spatially ordered sequence are called HoxD genes. They are controlled by an element called the GCR (global control region) in a manner very like that of LCR.

49. CHAPTER 17 Gene Regulation in eukaryotes 三、真核转录阻遏蛋白（或抑制蛋白）及其调控 Topic 3: Transcriptional Repressor & its regulation In eukaryotes, most repressors do not repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase. (Bacteria often do so)

50. Commonly, eukaryotic repressors recruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery (contrast to the activator recruited nucleosome modifers) histone deacetylases (组蛋白去乙酰化酶)removing the acetyl groups. Some modifier adds methyl groups to the histone tails, which frequently repress the transcription. This modification causes transcriptional silencing.