Lecture 6

1. Lecture 6 第3章 3.1 为什么使用RNA 3.2 转录机理 3.3 原核生物基因 表达调控 3.3.1 协同调控 3.3.2 乳糖操纵子 3.3.3 色氨酸操纵子 3.3.4 Ara与Gal 3.4 实验研究 Chapter 3 3.1 Why Use an RNA Intermediate? 3.2 Mechanism of Transcription 3.3 Regulation of Gene Expression in Prokaryotes 3.3.1 Coordinate Regulation 3.3.2 The Lac Operon 3.3.3 The Trp Operon 3.3.4 Ara & Gal Operons 3.4 Experiments

2. Review of Lecture 5 A1→ An RNA intermediate is used because … 使用RNA中间物是因为 ...... A2→ The player is RNA polymerase ! RNA聚合酶是玩家 ! A3→ Transcription is initiated at the promoter region. σ subunit of the RNA polymerase is responsible for recognizing the promoter. 转录是从启动子区域起始的，RNA 聚合酶的σ亚基负责识别启动子。 A4→ Transcription errors can be corrected in two ways 有两种方式可以用来纠正转录错误 A5→ Transcription can be terminated in two ways 转录能以两种方式终止

3. A1→ An RNA intermediate is used because … 使用RNA中间物是因为 ...... Chromosomes are large and complex 染色体庞大又复杂 DNA must be kept stableDNA必须保持稳定 One mRNA, many proteins 一条mRNA, 多个蛋白质 More opportunities for regulation 更多调控机会

4. A2→ The player is RNA polymerase ! RNA聚合酶是玩家 !

5. A3→ Transcription is initiated at the promoter region. σ subunit of the RNA polymerase is responsible for recognizing the promoter.转录是从启动子区域起始的，RNA聚合酶的σ亚基负责识别启动子。

6. A4→ Transcription errors can be corrected in two ways 有两种方式可以用来纠正转录错误

7. A5→ Transcription can be terminated in two ways 转录能以两种方式终止

8. Questions for Lecture 6 Q1→ How can E. coli survive when there is only lactose ? 只有乳糖时大肠杆菌如何生存呢？

9. Vocabulary of Lecture 6 (1/2) gene expression coordinate regulation polycistronic mRNA cistron operon operator lactose galactose permease galactosidase 基因表达 协同调控 多顺反子mRNA 顺反子 操纵子 操纵基因 乳糖 半乳糖 渗透酶 半乳糖苷酶

10. Vocabulary of Lecture 6 (2/2) transacetylase repressor catabolite activator protein adenylyl cyclase partial diploid negative regulation positive regulation allolactose mammalian gut 转乙酰基酶 阻遏蛋白 代谢物激活蛋白(CAP) 腺苷酸环化酶 部分二倍体 负调控 正调控 异乳糖 哺乳动物 肠，消化道

11. 3.3 Regulation of gene expression in prokaryotes3.3 原核生物基因表达调控 3.3.1 Coordinate Regulation 3.3.2 The Lac Operon 3.3.3 The Trp Operon 3.3.4 Ara & Gal Operons 3.3.1 协同调控 3.3.2 乳糖操纵子 3.3.3 色氨酸操纵子 3.3.4 阿拉伯糖与 半乳糖操纵子

12. Regulation of gene expression基因表达调控 Condition A

13. Regulation of gene expression基因表达调控 Condition B

14. 3.3.1 Coordinate Regulation协同调控 Operator 操纵基因 协同调控：一组基因在一起进行调控的转录调控方式。 Coordinate regulation:Transcriptional regulation in which a set of genes are regulated together.

15. Polycistronic mRNA多顺反子mRNA Polycistronic mRNA Translation Protein 1 Protein 3 Protein 2

16. 3.3.2 The lac Operon / 乳糖操纵子 lac操纵子：含有乳糖（lactose）代谢基因的操纵子。 Lac operon:An operon containing genes involved in lactose metabolism. The lac operon

17. Q1→ How can E. coli survive when there is only lactose ? 只有乳糖时大肠杆菌如何生存呢？ What will E. coli do with the lactose? Glucose 葡萄糖 Lactose 乳糖 E. coli: “I do not usually eat lactose, but in case when there is no glucose . . . ”

18. The Conditions of Lactose Metabolism乳糖代谢的条件 乳糖 葡萄糖 半乳糖 E. coli: “This is my favorite!”

19. How to use lactose / 如何利用乳糖 E. coli: “I need lactose permease, β-galactosidase, and transacetylase.”

20. When to use lactose / 何时利用乳糖 Glucose + Lactose + Glucose –Lactose + E. coli:“I will use lactose only when there is no glucose and there is lactose.” Glucose – Lactose –

21. Regulated Expression of Lactose Metabolizing Genes乳糖代谢基因表达调控 1. Negative Regulation – the lac Repressor 负调控──lac阻遏蛋白 2. Positive Regulation – CAP 正调控──CAP

22. Negative regulation and positive regulation负调控与正调控 负调控：关于转录，意思是当一种蛋白质结合上去以后引起转录的阻遏。 Negative regulation:With respect to transcription, means that the binding of a protein causes repression of transcription. 正调控：关于转录，表示通过一种蛋白质的结合而激活转录。 Positive regulation:In reference to transcription, denotes the activation of transcription by binding of a protein.

23. 1. Negative Regulation – When there is no lactose 负调控──当没有乳糖时

24. Negative Regulation – When there is lactose负调控──当有乳糖时

25. 2. Positive Regulation – CAP正调控──CAP 细胞能确保lac基因只在没有葡萄糖可以利用的情况下才表达

26. CAP-cAMP complex CAP CAP cAMP cAMP DNA cAMP的浓度与葡萄糖的浓度成反比

27. Adenylyl Cyclase (AC) O—CH2 A O O P——O OH O Glucose inhibits the activity of AC. ATP AC cAMP

28. CAP – “The accelerator” lac repressor – “The brake” Brake 刹车 Accelerator 加速器

29. A1→ Regulation of lac operon乳糖操纵子的调控

30. Now showingThe lac operon File: biophoto7 \ Genetics: A Conceptual Approach \ The lac operon (15’)

31. The lac operon (1/10) To utilize lactose as an energy source, E. coli must produce the enzymes necessary for its metabolism.

32. The lac operon (2/10) The genes for these enzymes are adjacent to one another on the bacterial chromosome and are transcribed together. A group of bacterial genes that are transcribed together is called an operon.

33. The lac operon (3/10) At one end of the lac operon is a set of structural genes, lacZ, lacY, and lacA, that encodes the enzymes necessary for metaboli-zing lactose. These three genes have a common promoter.

34. The lac operon (4/10) The regulator gene lacI is located upstream of the structural genes. The lacI gene encodes a repressor protein and is controlled by its own promoter. This repressor protein may bind to a region of the operon called the operator. The operator overlaps the 3’ end of the promoter and the 5’ end of the lacZ gene.

35. The lac operon (5/10) The lac operon is inducible—that is, the structural genes are expressed only in the presence of lactose. Active repressor proteins are encoded by the regulator gene. In the absence of lactose, these repressors bind to the operator and prevent RNA polymerase from initiating transcription.

36. The lac operon (6/10) When lactose is present, it is readily converted into allolactose, which may then come into contact with the repressor protein. Allolactose binds to the repressor protein and causes a conformational change that prevents the repressor from binding to the operator region of the DNA.

37. The lac operon (7/10) With the operator site open, RNA polymerase is able to bind to the operon promoter and trans-cribe the structural genes. The resulting mRNA is then translated, leading to the production of the three enzymes, β-galactosidase, premease, and transacetylase, encoded by lacZ, lacY, and lacA, respectively.

38. The lac operon (8/10) Lactose is a carbohydrate found in milk.It can be metabolized by E. coli bacteria that reside in the mammalian gut. Because lactose is too large to diffuse across the bacterial membrane, it must be actively transported into the cell by permease, encoded by the lacY gene of the lac operon.

39. The lac operon (9/10) To utilize lactose as an energy source, E. coli bacteria use the enzyme β-galactosidase, enco-ded by the lacZ gene of the lac operon, to break lactose down into glucose and galactose. The function in lactose metabolism of the third enzyme, transacetylase, aslo produced by the lac operon, is not known.

40. The lac operon (10/10) Here, we explore the effects of the lac repressor and operator genotypes in a partial diploid. Select one genotype for lacI and one genotype for lacO, and drag them to these places on the plasmid copy of the operon. The chromosome copy is fixed as wild type.

41. Now showing again

42. Now showingRegulation of Transcription Initiation File: Molecular Biology of the Gene \ Interactive animations Chapter 16 Regulation of Transcription Initiation. (~20’)

43. Essentials of Lecture 6 / 第6讲要点 A1→ Regulation of lac operon乳糖操纵子的调控

44. A1→ Regulation of lac operon乳糖操纵子的调控

45. Lecture 7 第3章 3.1 为什么使用RNA 3.2 转录机理 3.3 原核生物基因 表达调控 3.3.1 协同调控 3.3.2 乳糖操纵子 3.3.3 色氨酸操纵子 3.3.4 Ara与Gal 3.4 实验研究 Chapter 3 3.1 Why Use an RNA Intermediate? 3.2 Mechanism of Transcription 3.3 Regulation of Gene Expression in Prokaryotes 3.3.1 Coordinate Regulation 3.3.2 The Lac Operon 3.3.3 The Trp Operon 3.3.4 Ara & Gal Operons 3.4 Experiments

46. Questions for Lecture 7 Q1→ How should E. coli control tryptophan production? 大肠杆菌该如何控制色氨酸的生产呢？ Q2→E. coli: what should I do with the arabinose ?大肠杆菌：我应该如何对待阿拉伯糖呢？ Q3→E. coli: what should I do with the galactose ?大肠杆菌：我应该如何对待半乳糖呢？