The lac operon – An inducible operon

1. The lac operon – An inducible operon • Genes are either cis (coupling) acting • Genes affect those adjacent to themselves • Operator and structural genes Or • trans (repulsion) acting • Genes affect other genes that are not adjacent to themselves • Implies the action of a diffusible product • Regulator genes

2. Trans (repulsion) acting genes • Genes that affect other genes • Are not adjacent to the genes they control • Regulator genes Implies the action of a diffusible product

3. Genes that are cis (coupling) acting • Genes affect genes adjacent to themselves • Operator and structural genes

4. The lac operon – An inducible operon • Bacteria are monoploid – one copy of each gene • Can make bacteria partially diploid (two copies of a gene) • Place another copy of the gene(s) on a plasmid (F’) • E.coli lac I mutant with a plasmid carrying a functional repressor gene will be designated: I - O + Z +/F’ I +

5. The lac operon – An inducible operon

6. The lac operon – An inducible operon

7. The lac operon – An inducible operon

8. The lac operon – An inducible operon

9. The lac operon – An inducible operon

10. plasmid E. Coli genome

11. The lac operon – An inducible operon

12. The lac operon – An inducible operon

13. The lac operon – An inducible operon

14. The lac operon – An inducible operon In F’ plasmid, an entire lac operon can be supplied. For example, I + O + Z - Y +/F’ I - O + Z + Y - What are the beta-galactosidase and permease activities with and without lactose in the E. coli strain with the above genotype?

16. The lac operon • If E. coli cells are provided with both glucose and lactose, it preferentially metabolizes glucose rather than lactose. How do E. coli cells accomplish this? • Positive control and catabolite repression

17. Positive Control and Catabolite Repression New Players: Cyclic AMP (cAMP) CAP (catabolite activator protein)

18. Positive Control and Catabolite Repression High glucose low cAMP Low glucose high cAMP

19. Positive Control and Catabolite Repression High glucose low cAMP Low glucose high cAMP

20. Positive Control and Catabolite Repression High glucose low cAMP Low glucose high cAMP

21. The cAMP-CAP complex interacts with the promoter (neither can bind the promoter of lac operon by themselves) CAP = Catabolite Activator Protein

22. Facilitates the attachment of the RNA polymerase to the promoter – acts as an activator Must be a cAMP-CAP complex to bind to the promoter

23. Positive Control and Catabolite Repression What would happen to the lac operon when: • Glucose present (+), but lactose absent (-)

24. The cAMP-CAP complex is not formed (low cAMP), so CAP does not bind to the promoter (low) No transcription In addition, there is no inducer (lactose), so the active repressor is bound to the operator, and therefore the RNA polymerase cannot bind and transcribe the Lac operon genes

25. Positive Control and Catabolite Repression What would happen to the lac operon when: • Glucose present (+), but lactose absent (-) • No transcription

26. Positive Control and Catabolite Repression What would happen to the lac operon when: • Glucose present (+), but lactose absent (-) • No transcription • Glucose and lactose both present (+)

27. The cAMP-CAP complex does not form (low cAMP) • The inducer (lactose) is present and inactivates the repressor • There is some transcription, but it is inefficient (no cAMP-CAP complex)

28. Positive Control and Catabolite Repression What would happen to the lac operon when: • Glucose present (+), but lactose absent (-) • No transcription • Glucose and lactose both present (+) • Very little transcription

29. Positive Control and Catabolite Repression What would happen to the lac operon when: • Glucose present (+), but lactose absent (-) • No transcription • Glucose and lactose both present (+) • Very little transcription • Glucose absent (-), but lactose present (+)

30. The cAMP-CAP complex does form (high cAMP), facilitating the attachment of the RNA polymerase to the promoter The RNA polymerase is effectively attached to the promoter, enhancing transcription

31. Positive Control and Catabolite Repression What would happen to the lac operon when: • Glucose present (+), but lactose absent (-) • No transcription • Glucose and lactose both present (+) • Very little transcription • Glucose absent (-), but lactose present (+) • A lot of transcription

32. Positive Control and Catabolite Repression What would happen to the lac operon when: • Glucose present (+), but lactose absent (-) • No transcription • Glucose and lactose both present (+) • Very little transcription • Glucose absent (-), but lactose present (+) • A lot of transcription • Glucose and lactose both absent (-)

33. cAMP pairs with CAP and can attach to the promoter No inducer (no lactose)No transcription Repressor stays bound to the operator

34. Positive Control and Catabolite Repression What would happen to the lac operon when: • Glucose present (+), but lactose absent (-) • No transcription • Glucose and lactose both present (+) • Very little transcription • Glucose absent (-), but lactose present (+) • A lot of transcription • Glucose and lactose both absent (-) • No transcription

35. Lac operon overview - animation http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter12/animation_quiz_4.html