1 / 70

Scotty Merrell Department of Microbiology and Immunology B4140 dmerrell@usuhs.mil

Scotty Merrell Department of Microbiology and Immunology B4140 dmerrell@usuhs.mil Regulation of Gene Expression I. QUESTIONS. 1. Why does the expression of genes need to be regulated?. 2. Why is it important to study gene regulation?. 3. How is the expression of genes regulated?.

melosa
Download Presentation

Scotty Merrell Department of Microbiology and Immunology B4140 dmerrell@usuhs.mil

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Scotty Merrell Department of Microbiology and Immunology B4140 dmerrell@usuhs.mil Regulation of Gene Expression I

  2. QUESTIONS 1. Why does the expression of genes need to be regulated? 2. Why is it important to study gene regulation? 3. How is the expression of genes regulated? 4. How do we study gene regulation?

  3. Bacteria experience different conditions depending on environment Pathogenic bacteria: External reservoir Host Infection site #1 Infection site #2

  4. QUESTIONS 1. Why does the expression of genes need to be regulated? 2. Why is it important to study gene regulation? 3. How is the expression of genes regulated? 4. How do we study gene regulation?

  5. Pathogenic bacteria produce virulence factors when they sense they are inside of a host ICDDR,B Vibrio cholerae, the cause of cholera, produces toxin inside of the host. Understanding regulation of expression of this toxin is a means of understanding ways to prevent its production.

  6. QUESTIONS 1. Why does the expression of genes need to be regulated? 2. Why is it important to study gene regulation? 3. How is the expression of genes regulated? 4. How do we study gene regulation?

  7. Regulation of Gene Expression

  8. Regulation of Gene Expression

  9. RNA polymerase-promoter interactions Some promoters contain UP elements that stimulate transcription through direct interaction with the C-terminal domains of the  subunits of the RNA polymerase

  10. Arrangement of a  subunits on UP elements Promoter with a full UP element containing two consensus subsites. Promoter with an UP element containing only a consensus proximal subsite. Promoter with an UP element containing only a consensus distal subsite.

  11. Genes come in two main flavors: Constitutively expressed (transcription initiation is not regulated by accessory proteins) Regulated (transcription initiation is regulated by accessory proteins) a. Negatively Regulated--Repressor Protein b. Positively Regulated--Activator Protein

  12. Mechanisms of Regulation of Transcription Initiation: Negative Regulation RNA Polymerase

  13. Mechanisms of Regulation of Transcription Initiation: Negative Regulation Repressor Co-repressor Inactivator Repressor Repressor

  14. The lac operon a model for negative regulation A bacterium's prime source of food is glucose, since it does not have to be modified to enter the respiratory pathway. So if both glucose and lactose are around, the bacterium wants to turn off lactose metabolism in favor of glucose metabolism. There are sites upstream of the lac genes that respond to glucose concentration. This assortment of genes and their regulatory regions is called the lac operon.

  15. The lac promoter and operator regions

  16. The Lac Repressor is constitutively expressed Lac Repressor (monomer) (tetramer) Repressor binding prevents transcription

  17. When lactose is present, it acts as an inducer of the operon. It enters the cell and binds to the Lac repressor, inducing a conformational change that allows the repressor to fall off the DNA. Now the RNA polymerase is free to move along the DNA and RNA can be made from the three genes. Lactose can now be metabolized. Remember, the repressor acts as a tetramer

  18. When the inducer (lactose) is removed, the repressor returns to its original conformation and binds to the DNA, so that RNA polymerase can no longer get past the promoter to begin transcription. No RNA and no protein are made. Remember, the repressor acts as a tetramer

  19. How to identify the regulatory elements? 1. Mutation in the regulatory circuit may either abolish expression of the operon or cause it to occur without responding to regulation. 2. Two classes of mutants: A. Uninducible mutants: mutants cannot be expressed at all. B. Constitutive mutants: mutants continuously express genes that do not respond to regulation. 3. Operator (lacO): cis-acting element Repressor (lacI): trans-acting element

  20. Definitions: cis-configuration: description of two sites on the same DNA molecule (chromosome) or adjacent sites. cis dominance: the ability of a gene to affect genes next to it on the same DNA molecule (chromosome), regardless of the nature of the trans copy. Such mutations exert their effect, not because of altered products they encode, but because of a physical blockage or inhibition of RNA transcription. trans-configuration:description of two sites on different DNA molecules (chromosomes) or non-contiguous sites.

  21. Constitutive mutants: do not respond to regulation. Would this be a cis-dominant or recessive mutation?

  22. Constitutive mutants can be recessive

  23. Constitutive mutants can also be dominant if the mutant allele produces a “bad” subunit, which is not only itself unable to bind to operator DNA, but is also able to act as part of a tetramer to prevent any “good” (wild type LacI) subunits from binding. Pi lacI- O lacZ lacY lacA P X mRNA mRNA et al. mRNA lacI +

  24. Think about how you could determine whether a mutation was dominant or recessive.

  25. Questions about negative Regulation of lac ?

  26. Mechanisms of Regulation of Transcription Initiation: Positive Regulation RNA Polymerase

  27. Mechanisms of Regulation of Transcription Initiation: Positive Regulation RNA Polymerase Activator

  28. The lac operon a model for positive regulation When levels of glucose (a catabolite) in the cell are high, a molecule called cyclic AMP is inhibited from forming. So when glucose levels drop, more cAMP forms. cAMP binds to a protein called CAP (catabolite activator protein), which is then activated to bind to the CAP binding site. This activates transcription, perhaps by increasing the affinity of the site for RNA polymerase. This phenomenon is called catabolite repression, a misnomer since it involves activation, but understandable since when it was named, it seemed that the presence of glucose repressed all the other sugar metabolism operons.

  29. CAP --- a positive regulator 1. Catabolite repression: the decreased expression of many bacterial operons that results from addition of glucose. Also known as “glucose effect” or “glucose repression”. 2. E. coli catabolite gene activator protein (CAP; also known as CRP, the cAMP receptor protein). 3. CAP-cAMP activates more than 100 different promoters, including promoters required for utilization of alternative carbohydrate carbon sources such as lactose, galactose, arabinose, and maltose.

  30. IN OUT IIAGlc-P PTS Glucose Glucose-6-P IIAGlc How does glucose reduce cAMP level? PTS - phosphoenolpyruvate-dependent carbohydrate phosphotransferase system IIAGlc - glucose-specific IIA protein, one of the enzymes involved in glucose transport. 1. IIAGlc-P activates adenylate cyclase. 2. Glucose decreases IIAGlc-P level, thus reducing cAMP production. 3. Glucose also reduces CAP level: crp gene is auto-regulated by CAP-cAMP.

  31. Activation of expression of the lac operon

  32. E. coli CAP (CRP) --- 209 amino acids DNA-binding Helix-turn-helix Dimerization and cAMP-binding 1-139 140-209 -COOH NH2- AR1 156-164 His19 His21 Glu96 Lys101 AR2

  33. Transcription activation by CAP at class I CAP-dependent promoters (-62) • Transcription activation: • Interaction between the AR1 of the downstream CAP subunit and one copy of aCTD. • The AR1-aCTD interaction facilitates the binding of aCTD to the DNA downstream of CAP. • Possibly, interaction between same copy of aCTD and the s70bound at the –35 element. • 4. The interaction between the second aCTD and CAP is unclear. • The result: increasing the affinity of RNAP for promoter DNA, resulting in an • increase in the binding constant KB, for the formation of the RNAP-promoter closed complex

  34. Transcription activation by CAP at class I CAP-dependent promoters (cont.) (-103, -93, -83, or –72) Transcription activation: Possibly, the second copy of aCTD may interact with the DNA downstream of CAP, and may interact with the s70bound at the –35 element. Results: increasing the affinity of RNAP for promoter DNA, resulting in an increase in the binding constant KB, for the formation of the RNAP-promoter closed complex

  35. Transcription activation by CAP at class II CAP-dependent promoters (cont.) (-42) • Transcription activation: • Interaction between the AR1 of the upstream CAP subunit and one copy of aCTD • (either aCTDI or aCTDII, but preferentially aCTDI). The AR1-aCTD • interaction facilitates the binding of aCTD to the DNA upstream of CAP. • Results: increase in the binding constant KB, for the formation of the RNAP-promoter • closed complex • Interaction between the AR2 of the downstream CAP subunit and aNTDI. • Result: increase the rate constant, kf, for isomerization of closed complex to open complex.

  36. Transcription activation by CAP at class III CAP-dependent promoters (-103 or –93) (-62) Transcription activation: Each CAP dimer functions through a class I mechanism with AR1 of the downstream subunit of each CAP dimer interacting with one copy of aCTD Results: synergistic transcription activation

  37. Transcription activation by CAP at class III CAP-dependent promoters (cont.) (-42) (-103, -93, or -83) Transcription activation: The upstream CAP dimer functions by a class I mechanism, with AR1 of the downstream subunit interacting with one copy of aCTD; the downstream CAP dimer functions by a class II mechanism, with AR1 and AR2 interacting with the other copy of aCTD and aNTD, respectively. Results: synergistic transcription activation

  38. P Pi lacI O lacZ lacY lacA X mRNA Repressor Repressor monomer tetramer (c) No glucose (cAMP high); lactose present High level X of mRNA mRNA Inactive cAMP repressor CAP Repressor monomer Inducer High Repressor tetramer (b) Glucose present (cAMP low); lactose present (a) Glucose present (cAMP low); no lactose; No lactose inside the cells! (inducer exclusion)! Glucose effect on the E. colilac operon

  39. P Pi lacI O lacZ lacY lacA X mRNA Repressor Repressor monomer tetramer (c) No glucose (cAMP high); lactose present High level X of mRNA mRNA Inactive cAMP repressor CAP Repressor monomer Inducer High Repressor tetramer (b) Glucose present (cAMP low); lactose present (a) Glucose present (cAMP low); no lactose; No lactose inside the cells! (inducer exclusion)! Glucose effect on the E. colilac operon

  40. Inducer exclusion: How does it work? • Uptake of glucose dephosphorylates enzyme IIglc. • Dephosphorylated enzyme IIglc binds to and inhibits lactose permease. • Inhibition of lactose permease prevents lactose from entering the cell. • Hence, the term inducer exclusion.

  41. Questions about positive regulation of the lac operon?

  42. Dual positive and negative control of transcription initiation: the E. coliara operon

  43. The E. coli L-arabinose operon + +

  44. AraC exists in two states Arabinose P1 P2 Antiactivator Activator Arabinose

  45. AraC acts as a positive or negative regulator based on its conformational state and binding affinity for various sites in the two promoter regions. AraC encodes the regulator AraO1 and AraO2 encode operators CAP is a CAP binding site AraI is an additional regulatory region AraBAD are the structural genes

More Related