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 Ch 19: Comparative genomics and evolution of animal diversity

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 How genes are regulated to bestow cell type specificity in a group of genetically identical cells Chapter 19 How different animals diverse in genomes, why human is so special?

7. 6 Surfing the contents of Part IV

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 1-1 Gene Expression is Controlled by Regulatory Proteins 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 What are the transcription steps targeted by the regulators ?

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

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

18. 17 1-2 Targeting promoter binding: Many promoters are regulated by activators that help RNAP bind DNA and by repressors that block the binding  At many promoters, RNAP binds weakly  Lac operon is a good example Principles of Transcription Regulation

19. 18 a. Absence of Regulatory Proteins (operator) b. To Control Expression c. To Activate Expression Fig 16-1

20. 19 1-3 Targeting transition to the open complex: Some Activators Work by Allostery and Regulate Steps after RNA Polymerase Binding Fig 16-2 Examples: Activator promoter NtrCglnA MerRmerT

21. 20 Some promoters are inefficient at more than one step and can be activated by more than one mechanism Repressors can work in ways other than just blocking the promoter binding. For example, inhibition of the transition to the open complex.

22. 21 1-6 Targeting termination and beyond: Antitermination and Beyond  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. Principles of Transcription Regulation

23. 22 1-4 Action at a Distance and DNA Looping. Some proteins interact with each other even when bound to sites well separated on the DNA Fig 16-3

24. 23 Fig 16-4 DNA-binding protein can facilitate interaction between DNA- binding proteins at a distance Fig 16-4

25. 24 1-5 Cooperative Binding and Allostery have Many Roles in Gene Regulation  Cooperative binding: the activator interacts simultaneously with DNA and polymerase and so recruits the enzyme to the promoter  Group of regulators often bind DNA cooperatively: (1) produce sensitive switches to rapidly turn on a gene expression, (2) integrate signals (some genes are activated when multiple signals are present)

26. 25 Allostery is not only a mechanism of gene activation, it is also often the way that regulators are controlled by their specific signals.

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 5/10/2005

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 5/10/2005 Regulation of Transcription Initiation in Bacteria

31. 30 The lactose (Lac) Operon ( 乳糖操纵子 ) The enzymes required for the use of lactose as a carbon source are only synthesized when lactose is available as the sole carbon source. Fig 16-5 The LAC operon

32. 31 Lactose operon: a regulatory gene and 3 stuctural genes, and 2 control elements lacI Regulatory gene lacZ lacY lacA DNA m-RNA β -Galactosidase Permease Transacetylase Protein Structural Genes Cis-acting elements P lacI P lac O lac The LAC operon

33. 32 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

34. 33 1. The lacZ, lacY, lacA genes are transcribed into a single lacZYA mRNA (called polycistronic message) under the control of a signal promoter P lac. 2. 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

35. 34 2-1: An activator and a repressor together control the lac genes The activator: CAP (Catabolite Activator Protein) or CRP (cAMP Receptor Protein); responses to the glucose level. The repressor: lac repressor that is encoded by LacI gene; responses to the lactose. The LAC operon

36. 35 Fig 16-6 The LAC operon

37. 36 2-2: CAP and lac repressor have opposing effects on RNA polymerase binding to the lac promoter The LAC operon

38. 37 The site bound by lac repressor is called the lac operator. The LAC operon

39. 38 The lac operator overlaps promoter, and so repressor bound to the operator physically prevents RNA polymerase from binding to the promoter. Fig 16-8 The LAC operon

40. 39 CAP binds to a site with the similar structure as the operator, which is 60 bp upstream of the start site of transcription. CAP also interacts with the enzyme and recruit it to the promoter. Fig 16-9  CTD: C-terminal domain of the  subunit of RNAP

41. 40 2-3: CAP has separate activating and DNA- binding surface CAP binds as a dimer  CTD Fig 16-10 The LAC operon

42. 41 2-4: CAP and lac repressor bind DNA using a common structural motif The LAC operon

43. 42 Both CAP and lac repressor bind DNA using a helix-turn-helix motif. One is the recognition helix that can fits into the major groove of the DNA. Fig 16-11 The LAC operon

44. 43 DNA binding by a helix-turn-helix motif Fig 16-12 Hydrogen Bonds between repressor and the major groove of the operator The LAC operon

45. 44 Lac repressor binds as a tetramer, with each operator is contacted by a repressor dimer. In addition to the primary operator, there are two other lac operators located 400 bp downstream and 90 bp upstream, respectively. Not all the binding use a helix-turn-helix motif Fig 16-13

46. 45 2-5: The activity of Lac repressor and CAP are controlled allosterically by their signals Binding of the corresponding signals alter the structure of these two regulatory proteins The LAC operon

47. 46 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. Lactose is present, the low basal level of permease allows its uptake, andβ- 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

48. 47 Response to glucose The LAC operon

49. 48 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. 2-6: Combinatorial Control ( 组合调控 ): CAP controls other genes as well Regulation of Transcription Initiation in Bacteria

50. 49 Second example: Alternative  factor 5/10/2005 Regulation of Transcription Initiation in Bacteria

51. 50 2-7: Alternative  factor direct RNA polymerase to alternative site of promoters Alternative  factors

52. 51   factor:  factor subunit bound to RNA polymerase for transcription initiation

53. 52 Promoter recognition Different σfactors binding to the same RNA Pol Confer each of them a new promoter specificity  70 factors is most common one in E. coli under the normal growth condition Alternative  factors

54. 53 Many bacteria produce alternative sets of σfactors to meet the regulation requirements of transcription under normal and extreme growth condition E. coli : Heat shock  32 Sporulation in Bacillus subtilis Bacteriophage σfactors Alternative  factors

55. 54 Heat shock 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

56. 55 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

57. 56 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

58. 57 Normal bacterial holoenzyme Express early genes Encodeσfactor for transcription of late genes Encode σ 28 Express middle genes (gene 34 and 33 ) Alternative  factors

59. 58 Third example: NtrC and MerR and allosteric activation Third example: NtrC and MerR and allosteric activation 5/10/2005 Regulation of Transcription Initiation in Bacteria

60. 59 2-7: NtrC and MerR: Transcriptional activators that work by allostery rather than by recruitment NtrC and MerR and allosteric activation

61. 60 The majority of activators work by recruitment, such as CAP. These activators simply bring an active form of RNA polymerase to the promoter In this 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.

62. 61 In the absence of these activators, the 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

63. 62 2-8: 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, 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  NtrC phosphorylation and conformational change  The DNA binding domain binds DNA sites at ~ -150 position  NtrC interacts with  54 ( glnA promoter recognition)  ATP hydrolysis and conformation change in polymerase  transcription STARTs Fig 16-15 activation by NtrC

65. 64 2-8: 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, 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 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 undergo conformational change, which twists the promoter to restore it to the structure close to a strong  70 promoter

69. 68 2-9: Some repressors hold RNA polymerase at the promoter rather than excluding it Regulation of Transcription Initiation in Bacteria

70. 69 Repressors work in many ways : 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 Locking the promoter in a conformation incompatible with transcription initiation

71. 70 Fourth example: araBAD operon 5/10/2005 Regulation of Transcription Initiation in Bacteria

72. 71 2-10: AraC and control of the araBAD operon by antiactivation 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.

73. 72 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

74. 73 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 ?

75. 74 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 5/10/2005

76. 75 3-1: Amino acid biosynthetic operons are controlled by premature transcription termination: the tryptophan operon Examples of Gene Regulation at Steps After Transcription Initiation

77. 76 The trp operon encodes five structural genes required for tryptophan 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

78. 77 The Trp repressor （色氨酸阻遏物 ） The TRP operon

79. 78 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

80. 79 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 )

81. 80 trpR operon trp operon The TRP operon

82. 81 Attenuation ( 衰减子 ) : a regulation at the transcription termination step & a second mechanism to confirm that little tryptophan is available 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 The TRP operon

83. 82 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?)

84. 83 1. Transcription and translation in bacteria are coupled. 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.

85. 84 Fig 16-20 The leader RNA and leader peptide of the trp operon

86. 85 Complementary 3:4 termination of transcription Complementary 2:3 Elongation of transcription Four regions (1,2,3,4) of the leader sequence can base pair and form different structures depending on the ribosome action free leader RNA The TRP operon

87. 86 Low Trp High Trp Fig 16-21

88. 87 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.

89. 88 Box 4 Riboswitches Riboswitches are regulatory RNA elements that act as direct sensors of small molecule metabolites to control gene transcription or translation. Some operate at the level of transcription termination Others operate at the level of translation Another kind responds to the uncharged tRNA rather than responding to a metabolite. Antitermination mechanism.

90. 89

91. 90 3-2: Ribosomal proteins are translational repressors of their own synthesis: a negative feedback Examples of Gene Regulation at Steps After Transcription Initiation 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

92. 91 Strategies to meet the challenges: 1. Organization of the ribosomal proteins to several operons, 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 is at the level of translation, not transcription.

93. 92 Ribosomal protein operons The protein that acts as a translational repressor of the other proteins is shaded red. Fig 16-22

94. 93 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. Are proteins processed after translation? 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.

95. 94 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 regulated mRNA Fig 16-23

96. 95 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