1. Gene Regulation in Prokaryotes
Chapter 16
Gene Regulation
in Prokaryotes

2. Outline Part 1 Principles of Transcriptional Regulation
Part 2 Regulation of Transcription Initiation
Part 3 Examples of Gene Regulation after Transcription Initiation

3. Part 1 Principles of Transcriptional Regulation

4. 1-1 Gene Expression is Controlled by Regulatory Proteins
Genes are very often controlled by extracellular singals.The singals are communicatedto genes by regulateory proteins :
Postive regulators or activators
Increase the transcription
Negative regulators or repressors
Decrease or eliminates the transcription

5. 1-2 Many promoters are regulated by a ctivators that hel p RNAP bind DNA an d by repressors th at block the bindi ng

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

7. 1-3 Targeting transition to the open complex: Some Activators Work by Allostery and Regulate Steps after RNA Polymerase Binding
Fig 16-2

8. 1-4 Action at a Distance and DNA Looping
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

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

10. 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)

11. Part 2: Regulation of Transcription Initiation :
Examples from Bacteria

12. 2-1 Example from bacteria:Lac operon
The lactose (Lac) Operon (乳糖操纵子)

13. Lactose operon: a regulatory gene and 3 stuctural genes, and 2 control elements
Structural Genes
Cis-acting elements
DNA
lacI
lacZ
lacY
lacA
PlacI
Olac
Plac
m-RNA
Protein
β -Galactosidase
Transacetylase
Permease

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

15. An Activator and a Repressor Together Control the lac Genes
The activator is called CAP( Catabolite Activator Protein ) .CAP can bind DNA and activate the lac genes only in the absence of glucose.
The lac repressor can bind DNA and repress transcrition only in the absence of lactose.
Both CAP and lac repressor are DNA-binding proteins and each binds to a specific site n DNA at or near the lac promoter.

16. Fig 16-6

17. 2-2 CAP and lac repressor have opposing effects on RNA polymerase binding to the lac promoter
1.Lac operator the site bound by lac repressor
This 21 bp sequence is twofold summetric and is recognized by two subunits of lac repressor, one binding to each half-site.
Fig 16-7

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

19. 2-3 CAP has separate activating and DNA-binding surfaces
Fig 16-9
a CTD: C-terminal domain of the a subunit of RNAP

20. 2-4: CAP and lac repressor bind DNA using a common structural motif

21. Cap use the strucure called helix-turn-helix
The helix-turn-helix

22. lac repressor alse use the same mechanism
Fig Hydrogen Bonds between l repressor and the major groove of the operator

23. Cap and Lac repressor are differences in detail
Lac repressor binds as a tetramer not a dimer
Lac repressor ,other regions of protein ,outside the helix-turn-helix domain interact with the DNA.
In many cases ,binding of the protein does not alter the stricture of the DNA

24. The Difference
Lac repressor binds as a tetramer, with each operator is contacted by a repressor dimer.
Fig 16-13

25. 2-5: The activity of Lac repressor and CAP are controlled allosterically by their signals

26. Very low level of lac mRNA
Response to lactose i p o z y a
Very low level of lac mRNA
Absence of lactose
Active
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.
Presence of lactose i p o z y a
Inactive
Permease
b-Galactosidase
Transacetylase

27. Response to glucose

28. 2-6: Combinatorial Control (组合调控): CAP controls other genes as well
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.

29. EXAMPLE TWO---- ALTERNATIVE σFACTORS
2-7 Alternative s factor direct RNA polymerase to alternative site of promoters

30. Recall from Chapter 12 that it is the σsubunit of RNA polymerase that recognizes the promoter suquences.

31. Promoter recognition
Different σ factors bind to the RNA recognize the promoter sequence ,for example σ70. σ32

32. Third example: NtrC and MerR and allosteric activation
5/10/2005

33. 2-8 NtrC and Mert: Transcriptional Activators that Work by Allostery Rather than by Recruitment
NtrC controls expression of genes involved in nitrogen metabolism, such as the glnA gene. At the glnA gene, Ntrc induces a conformational change in the RNA Polymerase, triggering tansition to the open complex.
MerR controls a gene called merT. Like NtrC, MerR induces a conformational change in the inactive RNA polymerase-promoter complex, and this change can trigger open complex formation

34. Fig 16-15 activation by NtrC
NtrC Has ATPase Activity and Works from DNA Sites Far from the Gene
NtrC has separate activating and DNA-binding domains, and binds DNA when the nitrogen levels are low. The phosphorlated by a kinase. NtrC change the structure and display the activator domain
Fig activation by NtrC

35. The major process: Low nitrogen levels
Trigger polymerase to initiate transcription
NtrB phosphorylates NtrC
ATP hydrolysis and conformation change in polymerase
NtrC’s DNA-binding domain revealed
NtrC binds four sites located some 150 base pairs upstream of the promoter
NtrC interacts with 54

36. 2-9: MerR activates transcription by twisting promoter DNA

37. MerR bound to the single DNA-binding site, in the presence of mercury MerR activates the MerT gene. And the Mert twists the DNA.

38. Fig 16-15 Structure of a merT-like promoter

39. 2-10 Some repressors hold RNA polymerase at the promoter rather than excluding it

40. Repressors work in different ways
By binding to a site overlapping the promoter, it blocks RNA polymerase binding. (lac repressor)
The protein holds the promoter in a conformation incompatible with tanscription initiation.(the MerR case)
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)

41. Fourth example: araBAD operon

42. 2-11 AraC and control of the araBAD operon by antiactivation
The promoter of araBAD operon form E.coli is activated in the presence of arabinose and the absence of glucose and directs expression of gene encoding enzyme required for required for arabinose metabolism.

43. Figure 16-18 control of the araBAD operon
Different from the Lac operon, two activators AraC and CAP work together to activate the araBAD operon expression

44. Part three: Examples of gene regulation at steps after transcription initiation

45. 3-1 Amino acid biosynthetic operons are controlled by premature transcription termination
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.
Fig 16-19

46. The trp operon encodes five structural genes required for tryptophan synthesis.These genes are regulated to efficiently express only when tryptophan is limiting.There are two layers of regulation involved: (1) transcription repression by the Trp repressor (2) attenuation

47. The Trp repressor
When tryptophan is present, it binds the Trp repressor and induces a conformational change in that protein, enabling it to bind the trp operator and prevent transcription.When the tryptophan concentration is low, the Trp repressor is free of its corepressor and vacates its operator , allowing the synthesis of trp mRNA to commence from the adjacent promoter

48. Attenuation
a regulation at the transcription termination step & a second mechanism to confirm that little tryptophan is available

49. The using of the Repressor and Attenuation
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

50. The hairpin loop is followed by 8 uridine residues
The hairpin loop is followed by 8 uridine residues. At this so-called attenuator , transcription usually stops,yielding a leader RNA 139 nucleotides long
Figure trp operator leader RNA

51. 1 Transcription and translation in bacteria are coupled
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.

52. Figure 16-21 transcription at the trp attenuator

53. 3-2 Ribosomal Protein Are Translational Repressors of their Own Synthesis
Control of ribosome protein genes is simplified by their organization to several operons , each containing genes for up to 11 ribisomal proteins. Some nonribosomal proteins whose synthesis is also linked to growth rate are contained in these operons, including those for RNAP subunits a, b and b’. The primary control is at the level of translation, not transcription

54. Ribosomal protein operons

55. Ribosomal protein are repressors of their own translation
One ribosomal proteins binds the messenger near the translation initiation sequence of one the first genes in the operon ,preventing ribosomes from binding and initiating translation .repressing translation of the first genes also prevents expression of some or all of the rest.

56. How to overcome the challenges:
For each operon,one ribosomal protein binds the messenger near the translation initiation sequence of the first genes in the operon, preventing ribosomes from binding and initiating translation.
Repressing translation of the first gene also prevents expression of some or all of the rest.
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.

57. 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

58. Part four
The case of phage λ: layers of regulation

59. lysogeny
The alternative propagation pathway –involves integration of the phage DNA into the bacterial chromosome where it is passively replicated at each division –just as though it were a legitimate part of the bacterial genome

60. Lysogenic induction
When the cell is exposed to agents that damage DNA. This switch from Lysogenic to lytic growth is called lysogenic induction.

61. Lytic cycleand Establishment of lysogeny
Figure 16-24

62. 4-1 Alternative patterns of gene expression control lytic and lysogenic growth
Figure 16-25

63. Promoters in the right and left control regions of phage λ

64. Transcription in the λcontrol regions in lytic and lysogenic
Arrows indicate which promoters are active at the decisive period during lytic and lysogenic growth , respectively .the arrows also show the direction of transcription from each promoter

65. 4-2 Regulatory Proteins and Their Binding Sites
λrepressor, a protein of two domains joined by a flexible linker egion. λrepressor can both activate and repress trandcription Cro only represses transcription

66. 4-3 Repressor and Cro Bind in Different Patterns to Control Lytic and Lysogenic Growth
Repressor bound to OR1and OR2 turns off transcription from PR . And Repressor bound at OR2 contacts RNA polymerase at PRM, activating expression of the cI gene. OR3 lies with PRM; Cro bound there represses transcription of cI.

67. Relative positions of promoter and operator sites in OR

68. The action of λ repressor and Cro
Figure 16-31

69. Lysogenic induction requires proteolytic cleavage
Postiive autoregulation: when the level is too low the repressor activates its own repression.
Negative autoregulation: when the level is too high the repressor will bind to Or3 and repressing Rrm.

70. Negative autoregulation of repressor require long-distance interations and a large DNA loop

71. Another Activator, λcII, Controls the Decision between Lytic and Lysogenic Growth upon Infection of a new Host
cII is a transcriptional activator. It binds to a site upstream of a promoter called PRE and stimulates transcription of the cI gene from that promoter.

72. Interaction between the c-terminal domain of λ repressors
Figure 16-33

73. Growth conditions of E.coil control the stability of CII protein and thus/lysogenic choice

74. Transcriptional Antitermination in λ Development
The transcripts controlled by λN and Q proteins are initiated perfectly well in the absence of those regulators. But the transcripts terminate a few hundred to a thousand nucleotides downstream of the promoter unless RNA polymerase has been modified by the regulator; λN and Q protein are therefore called antiterminators.

75. Figure 16-36

76. Retoregulation:an interplay of control on RNA synthesis and stability determines int gene expression

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