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Chapter 16

Chapter 16. Gene Regulation in Prokaryotes. Outline. Part 1 Principles of Transcriptional Regulation. Part 2 Regulation of Transcription Initiation. Part 3 Examples of Gene Regulation after Transcription Initiation. Part 1 Principles of Transcriptional Regulation.

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Chapter 16

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  1. 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 1Principles 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 activators that help RNAP bind DNA and by repressors that block the binding

  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. 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 Regulatory gene Structural Genes Cis-acting elements DNA lacI lacZ lacY lacA PlacI Olac Plac m-RNA Protein Transacetylase β-Galactosidase Permease

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

  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 16-12 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. Response to lactose Absence of lactose z y a i p o Active Very low level of lac mRNA 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. Allolactoseacts as an inducer, binding to the lac repressor and inactivate it. Presence of lactose z y a i p o Inactive Permease Transacetylase b-Galactosidase

  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. 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 16-15 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. At this so-called attenuator , transcription usually stops,yielding a leader RNA 139 nucleotides long Figure 16-20 trp operator leader RNA

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