2. Welcome to MB Class

3. 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

4. The revised central dogma RNA processing Gene regulation 基因组的保持基因组的保持 基因组的表达基因组的表达 The structure of DNA and RNA

5. 4 Part IV Regulation Ch 16: Transcriptional regulation in prokaryotes Ch 17: Transcriptional regulation in eukaryotes Ch18: Regulatory RNAs Ch 19: Gene regulation in development and evolution Ch 20: Genome Analysis and Systems Biology

6. 5 Housekeeping genes: expressed constitutively, essential for basic processes involving in cell replication and growth. Inducible genes: expressed only when they are activated by inducers or cellular factors. Expression of many genes in cells are regulated

7. 6 Surfing the contents of Part IV --The heart of the frontier biological disciplines

8. 7 Some of the peoples who significantly contribute to the knowledge of gene regulation

9. 8

10. 9

11. 10 Chapter 16 Gene Regulation in Prokaryotes Chapter 16 Gene Regulation in Prokaryotes Molecular Biology Course

12. 11 TOPIC 1 Principles of Transcriptional Regulation [watch the animation] TOPIC 2 Regulation of Transcription Initiation: Examples from Bacteria (Lac operon, alternative  factors, NtrC,MerR, Gal rep, araBAD operon) TOPIC 3 The Case of Phage λ: Layers of Regulation

13. 12 Topic 1: Principles of Transcription Regulation CHAPTER 16 Gene Regulation in Prokaryotes 1.What are the regulatory proteins? 2.Which steps of gene expression to be targeted? 3.How to regulate? (recruitment, allostery, blocking, action at a distance, cooperative binding)

14. 13 1. Gene Expression is Controlled by Regulatory Proteins ( 调控蛋白 ) 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 Principles of Transcription Regulation

15. 14 2. Most activators and repressors act at the level of transcription initiation Principles of Transcription Regulation Why that? 1.Transcription initiation is the most energetically efficient step to regulate. [A wise decision at the beginning] 2.Regulation at this step is easier to do well than regulation of the translation initiation.

16. 15 Regulation also occurs at all stages after transcription initiation. Why? 1.Allows more inputs and multiple checkpoints. 2. The regulation at later stages allow a quicker response.

17. 16 Fig 12-3-initiation Promoter Binding (closed complex) Promoter “ melting ” (open complex) Promoter escape/Initial transcription

18. 17 Fig 12-3-Elongation and termination Termination Elongation

19. 18 recruitment block 3. Targeting promoter binding: Many promoters are regulated by activators ( 激活蛋白 ) that help RNAP bind DNA (recruitment) and by repressors ( 阻遏 蛋白 ) that block the binding. Principles of Transcription Regulation

20. 19 Generally, RNAP binds many promoters weakly. Why? Activators contain two binding sites to bind a DNA sequence and RNAP simultaneously, can therefore enhance the RNAP affinity with the promoters and increases gene transcription. This is called recruitment regulation ( 招募调控 ).*** On the contrary, Repressors can bind to the operator inside of the promoter region, which prevents RNAP binding and the transcription of the target gene.

21. 20 a. Absence of Regulatory Proteins: basal level expression b. Repressor binding to the operator represses expression c. Activator binding activates expression Fig 16-1

22. 21 4 Targeting transition to the open complex: Allostery regulation ( 异构调控 ) after the RNA Polymerase Binding Principles of Transcription Regulation In some cases, RNAP binds the promoters efficiently, but no spontaneous isomerization ( 异构化 ) occurs to lead to the open complex, resulting in no or low transcription. Some activators can bind to the closed complex, inducing conformational change in either RNAP or DNA promoter, which converts the closed complex to open complex and thus promotes the transcription. This is an example of allostery regulation.

23. 22 Fig 16-2 Allostery regulation Allostery is not only a mechanism of gene activation, it is also often the way that regulators are controlled by their specific signals.

24. 23 Repressors Repressors can work in ways: (1) blocking the promoter binding. (2) blocking the transition to the open complex. (3) blocking promoter escape

25. 24 5. Action at a Distance and DNA Looping. The regulator proteins can function even binding at a DNA site far away from the promoter region, through protein-protein interaction and DNA looping. Fig 16-3 Principles of Transcription Regulation

26. 25 Fig 16-4 DNA-binding protein can facilitate interaction between DNA- binding proteins at a distance Fig 16-4 Architectural protein

27. 26 6. Cooperative binding (recruitment) and allostery have many roles in gene regulation For example: group of regulators often bind DNA cooperatively (activators and/or repressors interact with each other and with the DNA, helping each other to bind near a gene they regulated) : (1) produce sensitive switches to rapidly turn on a gene expression. (1+1>2) (2) integrate signals (some genes are activated when multiple signals are present). Principles of Transcription Regulation

28. 27 Watch the animation-regulation of the transcription initiation!

29. 28 Topic 2: Regulation of Transcription Initiation : Examples from Bacteria Topic 2: Regulation of Transcription Initiation : Examples from Bacteria CHAPTER 16 Gene Regulation in Prokaryotes

30. 29 Operon: Operon: a unit of prokarytoic gene expression and regulation which typically includes: 1. Structural genes for enzymes in a specific biosynthetic and metabolic pathway whose expression is coordinately controlled. 2. Control elements, such as operator sequence. 3. Regulator gene(s) whose products recognize the control elements. These genes is usually transcribed from a different promoter.

31. 30 Control element Structural genes

32. 31 First example: Lac operon Regulation of Transcription Initiation in Bacteria The lactose Operon ( 乳糖操纵子 )

33. 32 Point 1: Composition of the Lac operon

34. 33 The enzymes encoded by lacZ, lacY, lacA are required for the use of lactose as a carbon source. These genes are only transcribed at a high level when lactose is available as the sole carbon source. Fig 16-5 The LAC operon 1. Lactose operon contains 3 structural genes and 2 control elements.

35. 34 lacY encodes a cell membrane protein called lactose permease ( 半乳糖苷渗透酶 ) to transport Lactose across the cell wall lacZ codes for β -galactosidase ( 半乳 糖苷酶 ) for lactose hydrolysis lacA encodes a thiogalactoside transacetylase ( 硫代半乳糖苷转 乙酰酶 ) to get rid of the toxic thiogalacosides The LAC operon

36. 35 polycistronic mRNA The lacZ, lacY, lacA genes are transcribed into a single lacZYA mRNA (polycistronic mRNA) under the control of a single promoter P lac. LacZYA transcription unit contains an operator site O lac position between bases -5 and +21 at the 3’-end of P lac Binds with the lac repressor The LAC operon

37. 36 Point 2: Regulatory proteins and their response to extracellular signals

38. 37 2. An activator and a repressor together control the Lac operon expression The activator: CAP (Catabolite Activator Protein, 代谢产物激活蛋白 ) or CRP (cAMP Receptor Protein, cAMP 受体蛋 白 ); responses to the glucose level. The repressor: lac repressor that is encoded by LacI gene; responses to the lactose. Sugar switch-off mechanism The LAC operon

39. 38 3. The activity of Lac repressor and CAP are controlled allosterically by their signals. Lactose is converted to allolactose by  - galactosidase, therefore lactose can indirectly turn off the repressor. Glucose lowers the cellular cAMP level, therefore, glucose indirectly turn off CAP. The LAC operon Allolactose binding: turn of Lac repressor cAMP binding: turn on CAP

40. 39 Fig 16-6 The LAC operon

41. 40 i i p p o o z z y y a a Very low level of lac mRNA Absence of lactose Active i p o z y a  -Galactosidase Permease Transacetylase Presence of lactose Inactiv e Lack of inducer: the lac repressor block all but a very low level of trans- cription of lacZYA. When Lactose is present, the low basal level of permease allows its uptake, and  -galactosidase catalyzes the conversion of some lactose to allolactose. Allolactose acts as an inducer, binding to the lac repressor and inactivate it. Response to lactose

42. 41 Response to glucose: 注意该图的 CRP 结合位点有误

43. 42 Point 3: The mechanism of the binding of regulatory proteins to their sites

44. 43 The LAC operon Repressor binding physically prevents RNAP from binding to the promoter Repressor binding physically prevents RNAP from binding to the promoter, because the site bound by lac repressor is called the lac operator (O lac ), and the O lac overlaps promoter (P lac ). 4. CAP and Lac repressor have opposing effects on RNA polymerase binding to the promoter The LAC operon

45. 44 The LAC operon CAP binds to a site upstream of the promoter, and helps RNA polymerase binds to the promoter by physically interacting with RNAP. CAP binds to a site upstream of the promoter, and helps RNA polymerase binds to the promoter by physically interacting with RNAP. This cooperative binding stabilizes the binding of polymerase to P lac. The LAC operon

46. 45 Fig 16-8 The LAC operon

47. 46 The LAC operon 5. CAP interacts with the CTD domain of the a-subunit of RNAP The LAC operon

48. 47 CAP site has the similar structure as the operator, which is 60 bp upstream of the start site of transcription. CAP interacts with the CTD domain of the  -subunit of RNAP and thus promotes the promoter binding by RNAP. Fig 16-9  CTD: C-terminal domain of the  subunit of RNAP

49. 48 CAP binds as a dimer  CTD Fig 16-10. CAP has separate activating and DNA-binding surface The LAC operon

50. 49 6. CAP and Lac repressor bind DNA using a common structural motif: helix-turn-helix motif The LAC operon Fig 16-11 One is the recognition helix that can fits into the major groove of the DNA. Another one sits across the major grove and makes contact with the DNA backbone.

51. 50 DNA binding by a helix-turn-helix motif Fig 16-12 Hydrogen Bonds between repressor and the major groove of the operator.

52. 51 Lac operon contains three operators: the primary operator and two other operators located 400 bp downstream and 90 bp upstream. Lac repressor binds as a tetramer ( 四聚体 ), with each operator is contacted by a repressor dimer ( 二聚体 ). respectively. Fig 16-13 DNA looping

53. 52 A regulator (CAP) works together with different repressors at different genes, this is an example of Combinatorial Control. In fact, CAP acts at more than 100 genes in E.coli, working with an array of partners. 7: Combinatorial Control ( 组合调 控 ): CAP controls other genes as well.

54. 53 Second example: Alternative  factor Regulation of Transcription Initiation in Bacteria Alternative  factors ( 可变  因子 ) direct RNA polymerase to alternative promoters.

55. 54  factor subunit bound to RNA polymerase for transcription initiation (Ch 12) Fig 12-7  and  subunits recruit RNA pol core enzyme to the promoter

56. 55 Different  factors binding to the same RNAP, conferring each of them a new promoter specificity.  70 factors is the most common one in E. coli under the normal growth condition.

57. 56 Many bacteria produce alternative sets of σfactors to meet the regulation requirements of transcription under normal and extreme growth condition. Bacteriophage has its own σfactors E. coli: Heat shock  32 Sporulation in Bacillus subtilis Bacteriophage σ factors

58. 57 Heat shock ( 热休克 ) Around 17 proteins are specifically expressed in E. coli when the temperature is increased above 37ºC. These proteins are expressed through transcription by RNA polymerase using an alternative  factor  32 coded by rhoH gene.  32 has its own specific promoter consensus sequences. Alternative  factors

59. 58 Many bacteriophages synthesize their own σfactors to endow the host RNA polymerase with a different promoter specificity and hence to selectively express their own phage genes. Bacteriophages Alternative  factors

60. 59 B. subtilis SPO1 phage expresses a cascade of σfactors which allow a defined sequence of expression of different phage genes. Fig 16-14 Alternative  factors

61. 60 Third example: NtrC and MerR use allosteric activation Third example: NtrC and MerR use allosteric activation Regulation of Transcription Initiation in Bacteria Transcriptional activators NtrC and MerR work by allostery rather than by recruitment.

62. 61 Review The majority of activators work by recruitment, such as CAP. These activators simply bring an active form of RNA polymerase to the promoter. The beautiful exceptions: allosteric activation by NtrC and MerR. In allosteric activation RNAP initially binds the promoter in an inactive complex, and the activator triggers an allosteric change in that complex to activate transcription.

63. 62 1. NtrC has ATPase activity and works at DNA sites far away from the gene. NtrC and MerR and allosteric activation NtrC controls expression of genes involved in nitrogen metabolism ( 氮代 谢 ), such as the glnA gene. NtrC has separate activating and DNA-binding domains, and binds DNA only when the nitrogen levels are low.

64. 63 Low nitrogen levels ( 低水平氮 )  NtrC phosphorylation and conformational change  NtrC (?) binds DNA sites at ~-150 bp position as a dimer (?)  NtrC interacts  54 in RNAP bound to the glnA promoter  NtrC ATPase activity provides energy needed to induce a conformation change in RNAP  transcription STARTs Fig 16-15 activation by NtrC

65. 64 2. MerR activates transcription by twisting promoter DNA NtrC and MerR and allosteric activation MerR controls a gene called merT, which encodes an enzyme that makes cells resistant to the toxic effects of mercury ( 抗汞酶 ) In the presence of mercury ( 汞 ), MerR binds to a sequence between –10 and –35 regions of the merT promoter and activates merT expression.

66. 65 As a  70 promoter, merT contains 19 bp between –10 and –35 elements (the typical length is 15-17 bp), leaving these two elements recognized by  70 neither optimally separated nor aligned.

67. 66 Fig 16-15 Structure of a merT-like promoter

68. 67 Fig 16-15 When Hg 2+ is absent, MerR binds to the promoter and locks it in the unfavorable conformation When Hg 2+ is present, MerR binds Hg 2+ and undergoes conformational change, which twists the promoter to restore it to the structure close to a strong  70 promoter

69. 68 Repressors work in many ways-review Blocking RNA polymerase binding through binding to a site overlapping the promoter. Lac repressor Blocking the transition from the closed to open complex. Repressors bind to sites beside a promoter, interact with polymerase bound at that promoter and inhibit initiation. E.coli Gal repressor Blocking the promoter escape. P4 protein interaction with P A2c (bacteriophage  29 )

70. 69 Fourth example: araBAD operon Regulation of Transcription Initiation in Bacteria

71. 70 1. AraC and control of the araBAD operon by anti-activation The araBAD operon The promoter of the araBAD operon from E. coli is activated in the presence of arabinose ( 阿拉伯糖 ) and the absence of glucose and directs expression of genes encoding enzymes required for arabinose metabolism. This is very similar to the Lac operon.

72. 71 Different from the Lac operon, two activators AraC and CAP work together to activate the araBAD operon expression Fig 16-18 CAP site 194 bp DNA looping

73. 72 Because the magnitude of induction of the araBAD promoter by arabinose is very large, the promoter is often used in expression vector. If fusing a gene to the araBAD promoter, the expression of the gene can be easily controlled by addition of arabinose （阿拉伯糖）. What is an expression vector ? [The answer is in the Methods part.]

74. 73 Topic 3: The Case of Bacteriophage : Layers of Regulation CHAPTER 16 Gene Regulation in Prokaryotes

75. Bacteriophage is a virus that infects E. coli. Upon infection, the phage can propagate in either of two ways : lytically or lysogenically. Bacteriophage is a virus that infects E. coli. Upon infection, the phage can propagate in either of two ways : lytically or lysogenically.

76. The phage has a 50-kb genome and ~50 genes. Most of these genes encode protein for replication, packing or lysis. How the lytic and lysogenic growth is regulated? ---regulatory proteins and cis-acting control elements.

77. 1.Alternative patterns of gene expression control lytic and lysogenic growth.

78. Fig. 16-21: Promoters in the right and left control regions of phage Fig. 16-21: Promoters in the right and left control regions of phage Fig. 16-22: Transcription in the  control regions in lytic and lysogenic growth

79. 2. Regulatory Proteins and Their Binding Sites The cI gene encodes repressor, that can both activate and repress transcription As a repressor: similarly as Lac repressor (?) As an activator: similarly as CAP (?)

80. repressor repressor As a repressor, it binds to sites that overlap the promoter and excludes RNA overlap the promoter and excludes RNA polymerase polymerase As an activator, it works like CAP by recruitment. recruitment. Cro (another regulatory protein), stands for control of repressor and other things. It is a single domain protein that binds as a dimer to 17-bp DNA sequences using a HTH motif. It only represses transcription.

81. repressor and Cro can each bind to any one of six operators, but with dramatically different affinity. repressor binds O R1 most easily while Cro binds O R3 with highest affinity.  repressor binds O R1 tenfold better than O R2. Cro binds O R3 tenfold better than O R1 and O R2. There are 6 operators in the right (3) and left (3) control regions of bacteriophage  Sequences are not identical

82. 3. repressor binds to operator sites cooperatively. Two dimmers of repressor bind cooperatively to O R1 and O R2, The binding at O R1 helps the binding at O R2. Monomer Dimer Tetramer High affinity 10-fold low affinity Not bound

83. Box 16-3 Concentration, affinity, and cooperative binding Two factors determine whether two interacting molecules find and bind each others: (1) the binding affinity (2) their concentrations. Cooperativity binding can be expressed in terms of increased affinity. The curve of l repressor binding to its operator DNA. Binding of a protein to a single site

84. 83 The benefit of cooperative binding of regulatory proteins is to ensure dramatic changes in the expression level of a given gene even in response to small changes in the level of the control signal.

85. Lytic growth Lysogen 4. Repressor and Cro bind in different patterns to control lytic and lysogenic growth

86. 1. DNA damage activates RecA in E. coli 2. RecA stimulates repressor to undergo autocleavage, resulting in the removal the C- terminal domain and the immediate loss of dimerization and binding cooperativity. 3. Repressor dissociates from O R1 -O R2 & O R1 -O R2, which triggers transcription from P R and P L 4. leading to lytic growth. 5. Lysogenic induction requires proteolytic cleavage of repressor

87. 86 For induction to work efficiently, the level of repressor in a lysogen must be tightly regulated. How?  Keep it not too low by positive autoregulation: repressor binding at O R2 activates its own transcription from P RM.  Keep it not too high by negative autoregulation: when the repressor level goes too high, it will bind to O R3 as well, which will prevents transcription from P RM.

88. When repressor level is high, it occupies both O R1 -O R2 and O L1 -O L2, and the interaction between two tetramer forms the repressor octomer and bring together O R3 and O L3 for another cooperative binding of the repressor. That’s why lysogeny can be so stable while also ensuring that induction is very efficient. 6. Negative autoregulation of repressor requires long-distance interactions and a large DNA loop: cooperative binding at O R3 and O L3. Fig. 16-27

89. Figure 16-28 Interactions between the c- terminal domain of repressors.

90. cII is transcribed from P R and cIII is transcribed from P L. CII protein is a transcriptional activator that binds to PRE and stimulate the transcription of cI gene ( repressor). 7. Another activator, CII, controls the decision between lytic and lysogenic growth upon infection of a new host-an earlier event.

91. Establishment of lysogeny: synthesis of the essential lysogenic repressor is established by transcription from one promoter and then maintained by transcription from another one.

92. 91 Establishment of lysogeny-a bigger view 1.P R and P L is constitutive promoters that promote transcription once the phage enter the cells. 2.P R directs the synthesis of both Cro and CII proteins. Cro favors lytic development while CII favors lysogentic growth by activating the synthesis of repressor. 3.The efficiency with which CII directs transcription of cI gene ( repressor) is critical in deciding the lysogeny. ??? What determines CII efficiency?

93. 8. The number of phage particles infecting a given cell affects whether the infection proceeds lytically or lysogenically. When more CII proteins are made from more infected phages, there is a larger chance to produce enough repressor to produce lysogeny. 9. Growth conditions of E. coli control the stability of CII protein and thus the lytic/lysogenic choice. Infection of Healthy and growing vigorously bacterial cells >>CII is unstable >>propagates lytically because. When conditions are poor for bacterial growth >>CII becomes stable >> form lysogens and sit tight. [CII is degraded by a specific protease FtsH]

94. 10. Transcriptional Antitermination in development: examples of regulation after transcription initiation. Two phage regulatory proteins N and Q, called antiterminators, prevent the termination at some termination sites and promotes the transcription of the early late and late genes for the lytic growth of the phage.

95. Gene expression in lytic growth Three phases: Immediate early: Transcription starts at P R and P L that flank the cI and stops at the  - dependent terminators (t) after the N and cro genes; Delayed early: Transcription begins at the same promoters, but bypasses the terminators by virtue of the N gene product, N, which is an antiterminator; Late: Transcription begins at a new promoter P’ R ; it would stop short at the t without the Q gene product, Q, another antiterminator.

96. 95 N proteins binds to the RNA Q proteins bind to the QBE DNA site.

97. Antitermination by N protein The gene surrounding N are depicted along with the leftward promoter (P L ) and operator (O L ), the terminator and the nut site. The gene surrounding N are depicted along with the leftward promoter (P L ) and operator (O L ), the terminator and the nut site. Transcription in the absence of N Transcription in the absence of N Transcription in the presence of N Transcription in the presence of N

98. 97 1.Principles of gene regulation. (1) who regulate? (2) where to target? (3) How to regulate? 2.Regulation of transcription initiation in bacteria: the lac operon, alternative  factors, NtrC, MerR, araBAD operon. 3.The case of phage--layers of regulation:  repressor and Cro and their binding; control of the lytic and lysogenic growth; lysogenic induction; control of the decision to lytic or lysogenic growth by  CII; Antiterminators. Key points of the chapter