Molecular Mechanisms of Gene Regulation: The Operon (Ch7)

1. Molecular Mechanisms of Gene Regulation: The Operon (Ch7)

2. Operon- set of genes that are coordinately controlled by a regulatory protein AND transcribed as a single polycistronic message Regulon- set of related genes that are transcribed as separate units but are controlled by the same regulatory protein

3. The Lactose Operon lacZ : b-galactosidase lacY : lactose (galactoside) permease lacA : galactoside transacetylase

4. Diauxic growth Bi-phasic; cells grow on one carbon source until depleted & then grow on the other

5. Francois Jacob Jaques Monod

6. 1. Diauxic growth is dependent upon the carbon (sugar) source used. 2. In E. coli: two classes of sugar sources (i) glucose, mannose, fructose (ii) lactose, maltose 3. Growth on class (i) combinations, i.e. glucose + mannose  no diauxic growth; same with class (ii) mixtures. 4. Diauxy is observed when cells are grown in mixtures containing (i) + (ii).

7. Induction of the lac operon

8. Negative Regulation of transcription Inducible

9. Negative Regulation Repressible

10. Positive Regulation

12. The lac Operon

13. The nature of the lac inducer

14. Complementation • Restoration of phenotype 2. Different types: genetic material 3. Mutation with phenotype  add DNA (gene product)  restores phenotype Typical conclusion: mutation & complementing DNA encode-for or are the same gene Alternate conclusions: compensatory affects

15. Complementation using two (recessive) mutants Interpretations  very different

17. Mutant Repressor Gene Lac product? + inducer - inducer (no repressor made) Y/N Y/N Y/N Y/N Conclusion: Both lac operons are repressible recessive

18. Mutant Operator (Oc) Lac product? Y/N Y/N Conclusion: One lac operon non-repressible cis-dominant

19. Mutant Repressor Gene (cannot bind inducer) Lac product? Y/N Y/N Conclusion: Both lac operons are uninducible cis and trans dominant

20. Mutant Repressor Gene Lac product? (cannot bind operator sequence) Y/N Y/N Conclusion: Both lac operons are non-repressible dominant-negative

21. Repression & Activation

22. Binding between lac Operator & lac Repressor

23. Non-metabolizable analogue of lactose

24. The lac control region 1. 3 operators (O1, O2, O3); region where regulatory proteins bind 2. RNA polymerase binding site (promoter) 3. cAMP-CRP complex binding site (CAP)

25. b-Galactosidase Activity 1. Recall that the first gene in the lac operon is lacZ (b-galactosidase) 2. Enzyme activity can easily be measured using X-Gal or p-nitrophenol-galactoside (colorimetric assays that can be quantified) 3. Therefore effects on regulation can be monitored by measuring b-galactosidase activity.

26. Effects of Mutations in the 3 lac Operators

27. Positive Control of the lac Operon 1. Removal of repressor is NOT enough to activate the operon. 2. The lac operon has a mechanism for reponding to glucose levels. Why? – (i) When glucose levels are high, the cell wants to repress transcription of other operons (lactose) (ii) When glucose levels are low & lactose present  upregulate lac operon  Catabolite repression selection in favor of glucose metabolism

28. -cAMP responds to glucose conc. ATP Inhibited by glucose Adenylcyclase - glucose uptake lowers the quantity of cAMP by inhibiting the enzyme adenylcyclase. Cyclic AMP

29. 1. Addition of cAMP overcomes catabolite repression. 2. The activator is a complex between cAMP and a protein: catabolite activator protein (CAP) aka cAMP receptor protein (CRP)  gene crp. 3. A mutant CRP protein with 10 lower affinity for cAMP: if cAMP-CRP complex important for activation then mutant should have reduced production of b-galactosidase

31. The Molecular Mechanism of c-AMP-CRP Action

32. 1. cAMP-CRP complex stimulates transcription by binding to (activator) site adjacent to promoter. 2. cAMP-CRP recruits and helps RNA polymerase to bind to the promoter. 3. Recruitment has two steps: -formation of closed promoter complex -conversion of closed promoter complex to open promoter complex increases rate of open promoter complex formation

33. Rifampicin-inhibits RNA polymerase Only if added before RNA polyermase has initiated transcription  rifampicin resistant complex + rifampicin + nucleotides

34. + rifampicin + nucleotides Conclusion- cAMP-CRP (CAP) promotes open promoter complex formation

35. How does cAMP-CRP binding to the activator site facilitate binding of polymerase to the promoter? 1. cAMP-CRP complex “touches” the polymerase  cooperative binding 2. cAMP-CRP causes the DNA to bend.

36. Direct Interaction Model Evidence: (1) co-sedimentation (2) chemical cross-linking (3) Dnase footprinting (4) mutations in CRP that decrease activation but NOT DNA binding  interface that interacts with polymerase.

37. DNA Looping -cooperative binding between proteins to remote sites

38. Measuring DNA bending 1. cut DNA fragment with different restriction enzymes

39. 2. Bind protein

40. Relationship between electrphoretic mobility and bent DNA (w/protein) Bend center  protein binding site

41. DNA bending model for cAMP-CRP activation -bend facilitates polymerase binding (exposes promoter)

42. Mechanism of Repression 1. Assumption: repressor blocks polymerase access to promoter. 2. Experimental evidence, however, has shown that RNA polymerase can STILL bind to promoter in the presence of repressor Rifampicinno transcription unless open promoter complex has formed Experiment 1: DNA, polymerase, repressor  add inducer, nucleotides, & rifampicin Result : Transcription occurred  repressor had not prevented formation of open complex

43. Experiment 2: 1. DNA + repressor (5-10 min) 2. + RNA polymerase (20 min) 3. Add heparin -Blocks any further complex formation + all reaction components except CTP 4. Add CTP +/- inducer (IPTG)

44. -sulfated glycosoaminoglycan (chain) -joints, vitreous humor -viscosity increasing agent, anti-coagulant -binds RNA polymerase inhibiting association with promoter

45. Further evidence showed that repressor and polymerase can bind together to lac operator. If lac repressor does not inhibit transcription of the lac operon by blocking access to promoter, how does it function? Alternate theory: repressor locks RNA polymerase into a non-productive state. Evidence: formation of abortive transcripts

46. HOWEVER… More recent studies have shown that repressor/polymerase : operator interactions are in equilibrium. Ratio of: polymerase-promoter complex and free polymerase/free promoter And that previous experiments were simply shifting or locking this equilibrium association

47. Experiment: 1. Add RNA polymerase + lac promoter (used fluorescent labeled UTP analog) • (1) no addition (2) + heparin • (3) + repressor (4) no DNA Analysis: (i) heparin known to prevent polymerase (re)-association (ii) If repressor does not block access to polymerase it should not inhibit polymerase association with promoter

49. Result: both heparin and repressor inhibits (re)-association of polymerase with promoter. Analysis: (1) heparin binds polymerase preventing association with DNA (2) repressor does the same by binding to the operator adjacent to the promoter and blocking access to the promoter by RNA polymerase. Conclusion: Original competition hypothesis may be correct!