2. Welcome Each of You to My Molecular Biology 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. 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)

5. 4 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

6. 5 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

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 Examples of Gene Regulation after Transcription Initiation (Trp operon) TOPIC 4 The Case of Phage λ: Layers of Regulation

13. 12 Topic 1: Principles of Transcription Regulation CHAPTER 16 Gene Regulation in Prokaryotes 5/10/2005

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. Gene expression is controlled at different stages ( 基因表达可以发生 在不同时期 ) Principles of Transcription Regulation  The bulk of gene regulation takes place at the initiation of transcription.  Some involve transcriptional elongation/termination, RNA processing, and translation of the mRNA into protein.

16. 15 Fig 12-3-initiation Promoter Binding (closed complex) Promoter “ melting ” (open complex) Promoter escape/Initial transcription

17. 16 Fig 12-3-Elongation and termination Termination Elongation

18. 17 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. RNAP binds many promoters weakly (?), activators that contain two binding sites to bind a DNA sequence and RNAP simultaneously can enhance the RNAP affinity with the promoters, and thus 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. Principles of Transcription Regulation

19. 18 a. Absence of Regulatory Proteins: basal level expression b. Repressor binding to the operator represses expression c. Activator binding activates expression Fig 16-1

20. 19 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 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.

21. 20 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.

22. 21 5 Targeting promoter escape by some repressors Principles of Transcription Regulation Repressors can work in ways: (1) blocking the promoter binding. (2) blocking the transition to the open complex. (3) blocking promoter escape

23. 22 Some promoters are inefficient at more than one step and can be activated by more than one mechanism. Activation mechanisms include recruitment ( 招募 ) and allostery ( 异构 ).

24. 23 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, (2)integrate signals (some genes are activated when multiple signals are present). Principles of Transcription Regulation

25. 24 7. 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

27. 26 Topic 2: Regulation of Transcription Initiation : Examples from Bacteria Topic 2: Regulation of Transcription Initiation : Examples from Bacteria CHAPTER 16 Gene Regulation in Prokaryotes

28. 27 Operon: Operon: a unit of prokarytoic gene expression and regulation which typically includes: 1. Structural genes for enzymes in a specific biosynthetic pathway whose expression is coordinately controlled. 2. Control elements, such as operator sequence. 3. Regulator gene(s) whose products recognize the control elements. Sometimes are encoded by the gene under the control of a different promoter

29. 28 Control element Structural genes

30. 29 First example: Lac operon Regulation of Transcription Initiation in Bacteria The lactose Operon ( 乳糖操纵子 )

31. 30 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 a regulatory gene and 3 structural genes, and 2 control elements.

32. 31 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

33. 32 The lacZ, lacY, lacA genes are transcribed into a single lacZYA mRNA (polycistronic mRNA) under the control of a signal 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

34. 33 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

35. 34 Fig 16-6 The LAC operon

36. 35 The LAC operon The site bound by lac repressor is called the lac operator (O lac ), and the O lac overlaps promoter (P lac ). Therefore repressor bound to the operator physically prevents RNA polymerase from binding to the promoter. 3. Lac repressor bound to the operator prevents RNAP from binding to the promoter The LAC operon

37. 36 Fig 16-8 The LAC operon

38. 37 The LAC operon CAP has two binding sites, one interacts with the CAP site on the DNA near promoter, and one interacts with RNAP. This cooperative binding ensures that RNAP effectively binds to P lac and initiates transcription of LacZYA. 4. CAP activates the Lac transcription through recruitment of RNAP to the weak P lac The LAC operon

39. 38 CAP site has the similar structure as the operator, which is 60 bp upstream of the start site of transcription. CAP also interacts with the RNAP and recruit it to the promoter. Fig 16-9  CTD: C-terminal domain of the  subunit of RNAP

40. 39 CAP binds as a dimer  CTD Fig 16-10. CAP has separate activating and DNA-binding surface The LAC operon

41. 40 5. 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.

42. 41 DNA binding by a helix-turn-helix motif Fig 16-12 Hydrogen Bonds between repressor and the major groove of the operator.

43. 42 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

44. 43 6 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: turn of Lac repressor cAMP: turn on CAP

45. 44 i p o z y 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

46. 45 Response to glucose

47. 46 A regulator (CAP) works together with different repressor 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

48. 47 Second example: Alternative  factor Regulation of Transcription Initiation in Bacteria Alternative  factor ( 可变  因子 ) direct RNA polymerase to alternative site of promoters

49. 48  factor subunit bound to RNA polymerase for transcription initiation (Ch 12)

50. 49 Different  factors binding to the same RNAP, conferring each of them a new promoter specificity.  70 factors is most common one in E. coli under the normal growth condition

51. 50 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 σfactors

52. 51 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

53. 52 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

54. 53 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

55. 54 Third example: NtrC and MerR and allosteric activation Third example: NtrC and MerR and allosteric activation Regulation of Transcription Initiation in Bacteria Transcriptional activators NtrC and MerR work by allostery rather than by recruitment.

56. 55 Review The majority of activators work by recruitment, such as CAP. These activators simply bring an active form of RNA polymerase to the promoter In the case of allosteric activation, RNAP initially binds the promoter in an inactive complex, and the activator triggers an allosteric change in that complex to activate transcription.

57. 56 In the absence of NtrC and MerR, RNAP binds to the corresponding promoter to form a closed stable complex. NtrC activator induces a conformational change in the enzyme, triggering transition to the open complex MerR activator causes the allosteric effect on the DNA and triggers the transition to the open complex

58. 57 1. NtrC has ATPase activity and works from DNA sites far 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.

59. 58 Low nitrogen levels ( 低水平氮 )  NtrC phosphorylation and conformational change  NtrC (?) binds DNA sites at ~-150 positio as a dimer  NtrC (?) interacts with  54 ( glnA promoter recognition)  NtrC ATPase activity provides energy needed to induce a conformation change in polymerase  transcription STARTs Fig 16-15 activation by NtrC

60. 59 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.

61. 60 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.

62. 61 Fig 16-15 Structure of a merT-like promoter

63. 62 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

64. 63 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 )

65. 64 Fourth example: araBAD operon Regulation of Transcription Initiation in Bacteria

66. 65 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.

67. 66 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

68. 67 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.]

69. 68 Topic 3: Examples of Gene Regulation at Steps After Transcription Initiation Topic 3: Examples of Gene Regulation at Steps After Transcription Initiation CHAPTER 16 Gene Regulation in Prokaryotes

70. 69 First example: the tryptophan operon ( 色 胺酸操纵子 ) Examples of Gene Regulation at Steps After Transcription Initiation

71. 70 1. Amino acid biosynthetic operons are controlled by premature transcription termination: the trp operon

72. 71 The trp operon encodes five structural genes required for tryptophan ( 色胺酸 ) synthesis. These genes are regulated to efficiently express only when tryptophan is limiting. Two layers of regulation are involved: (1) transcription repression by the Trp repressor (initiation); (2) attenuation The TRP operon

73. 72 The Trp repressor （色氨酸阻遏物 ） The TRP operon

74. 73 1. Trp repressor is encoded by a separate operon trpR, and specifically interacts with the operator that overlaps with the promoter sequence 2. The repressor can only bind to the operator when it is complexed with tryptophan. Therefore, Try is a co- repressor and inhibits its own synthesis through end-product inhibition (negative feed-back regulation). The TRP operon Remember the lac repressor acts as an inducer

75. 74 The TRP operon 3. The repressor reduces transcription initiation by around 70-fold, which is much smaller than the binding of lac repressor. 4. The repressor is a dimer of two subunits which has a structure with a central core and two flexible DNA-reading heads (carboxyl-terminal of each subunit )

76. 75 trpR operon trp operon The TRP operon

77. 76 Attenuation ( 衰减作用 ) : a regulation at the transcription termination step & a second mechanism to confirm that little tryptophan is available The TRP operon

78. 77 Repressor serves as the primary switch to regulate the expression of genes in the trp operon Attenuation serves as the fine switch to determine if the genes need to be efficiently expressed

79. 78 Fig 16-19 Transcription of the trp operon is prematurally stopped if the tryptophan level is not low enough, which results in the production of a leader RNA of 161 nt. (WHY?)

80. 79 1. Transcription and translation in bacteria are coupled ( 细菌体内的转录和翻译是偶联的 ). Therefore, synthesis of the leader peptide immediately follows the transcription of leader RNA. 2. The leader peptide contains two tryptophan codons. If the tryptophan level is very low, the ribosome will pause at these sites. 3. Ribosome pause at these sites alter the secondary structure of the leader RNA, which eliminates the intrinsic terminator structure and allow the successful transcription of the trp operon.

81. 80 Fig 16-20 The leader RNA and leader peptide of the trp operon

82. 81 Low Trp High Trp Fig 16-21 Complementary 2:3 Elongation of transcription Complementary 3:4 termination of transcription

83. 82 Importance of attenuation 1. A typical negative feed-back regulation 2. Use of both repression and attenuation allows a fine tuning of the level of the intracellular tryptophan. 3. Attenuation alone can provide robust regulation: other amino acids operons like his and leu have no repressors and rely entirely on attenuation for their regulation. 4. Provides an example of regulation without the use of a regulatory protein, but using RNA structure instead.

84. 83 Second example: Riboswitches- a RNA structure control mechanism Examples of Gene Regulation at Steps After Transcription Initiation Riboswitches are regulatory RNA elements that act as direct sensors of small molecule metabolites to control gene transcription or translation.

85. 84 Box 4 1.Riboswitches operating at the level of transcription termination using an Antitermination mechanism. 2.Riboswitches operating at the level of translation, controlling the formation of an RNA structure that masks the ribosome binding site on mRNA.

86. 85 Tucker1 and Breaker, Current Opinion in Structural Biology 2005, 15:342–348 代谢物

87. 86 The 2nd structures of 7 riboswitches and metabolites that they sense

88. 87 Third example: Ribosomal proteins are translational repressors of their own synthesis: a negative feedback Examples of Gene Regulation at Steps After Transcription Initiation

89. 88 Challenges the ribosome protein synthesis 1. Each ribosome contains some 50 distinct proteins that must be made at the same rate. 2. The rate of the ribosome protein synthesis is tightly closed to the cell’s growth rate.

90. 89 Strategies to meet the challenges-Operon 1. Organization of the ribosomal proteins to several operons ( 操纵子 ), each containing up to 11 ribosomal protein genes 2. Some nonribosomal proteins whose synthesis is also linked to growth rate are contained in these operons, including those for RNAP subunits ,  and  ’. 3. The primary control ( 主要调控 ) is at the level of translation, not transcription.

91. 90 Ribosomal protein operons The protein that acts as a translational repressor of the other proteins is shaded red. Fig 16-22

92. 91 Strategies to meet the challenges (cont): 4. For each operon, one (or two) ribosomal proteins binds the mRNA near the translation initiation sequence, preventing the ribosome from binding and initiating translation. 5. Repressing translation of the first gene also prevents expression of some or all of the rest. 6. The strategy is very sensitive. A few unused molecule of protein L4, for example, will shut down synthesis of that protein and other proteins in this operon.

93. 92 7. The mechanism of one ribosomal protein also functions as a regulator of its own translation: the protein binds to the similar sites on the ribosomal RNA and to the regulatory RNA in its own mRNA. Fig 16-23

94. 93 1.Principles of gene regulation. (1) The targeted gene expression events; (2) the mechanisms: by recruitment/exclusion or allostery 2.Regulation of transcription initiation in bacteria: the lac operon, alternative  factors, NtrC, MerR, Gal rep, araBAD operon 3.Examples of gene regulation after transcription initiation: the trp operon, riboswitch, regulation of the synthesis of ribosomal proteins Key points of the chapter