1. Distinguish between positive and negative transcriptional regulation Make predictions based on hypotheses Infer protein function from mutant phenotype Pages 519-525 of textbook Transcriptional Regulation of the L-ara operon Originally written by Lazar Dimitrov

2. Example of catabolite control 1.Grow cells in minimal media with lactose as the carbon course 2. Add Glucose 3. Measure Beta-galactosidase-LacZ -Cell default setting is to use glucose for energy production

3. lactose galactose glycerol maltose sorbose Sugar Utilized

4. PEP (phospho enol pyruvate)-dependent sugar phosphotransferase system-transports glucose into the cells -PTS IIA glc exists in two form +/- phosphate -phospho IIA glc activates adenylate cyclase -ration of IIA glc to IIA glc -P depends on glucose availability -Hpr Histidine protein adds phosphates Glucose Glycolysis PEP:Pyruvate TCA Cycle Regulation of cAMP Production

5. The arabinose regulon

6. Figure 12.18

7. Mutant Analysis Lactose Galactose & Glucose lacY, lacZ Glycolysi s Negative Regulation (lacI) Is it subject to positive or negative regulation? L- arabinose L-ribulose L-ribulose- 5- phosphate D-xylulose- 5- phosphate L-arabinose Utilization

8. How Many Genes? Mutagenesi s Minimal Glucose Minimal L- arabinose Replica plate Ara - mutants Test each Ara - for dominance/recessivity to w.t. Set up complementation tests between all possible recessive Ara - mutant pairs

9. How Many Genes? At least 4 complementation groups/genes (araA, araB, araC and araD) are defined by the Ara - mutants L- arabinose L-ribulose L-ribulose- 5- phosphate D-xylulose- 5- phosphate araAaraBaraD What about araC

10. araC - Mutants Are “Super- suppressed” C-C- BAD OFF No L-arabinose Plus L- arabinose What could be the function of araC? (Hint: araC is not the L-ara permease) C-C- BAD

11. C-C- BAD OFF C-C- BAD No L- arabinose Plus L-arabinose Is araC a Positive or Negative Regulator? I-I- ZYA ON No Lactose I-I- ZYA ON Plus Lactose

12. Is araC a Positive or Negative Regulator? What mutations in the lac operon cause “super- suppressed” phenotype?

13. Negative Regulation Wild Type Plus inducer Gene YPromoter RNAPol ON Gene YPromoter Wild type No inducer OFF Constitutive Mutants Gene YPromoter RNAPol ON Loss-of-function (LOF) mutations

14. Negative Regulation & Constitutive Mutants No inducer - Gene will be ON/OFF? Gene YPromoter

15. Negative Regulation & Constitutive Mutants Plus inducer - Gene will be ON/OFF? Gene YPromoter

16. Partial diploid containing a constitutive allele and a wild type allele has wild type phenotype THEREFORE, constitutive mutants are dominant/recessive to wild type under negative regulation Negative Regulation & Constitutive Mutants

17. Positive Regulation Gene YPromoter Wild type No inducer Wild Type Plus inducer Gene YPromoter RNAPol ON OFF Constitutive Mutants Gene YPromoter RNAPol ON Gain-of-function (GOF) mutations

18. Positive Regulation & Constitutive Mutants No inducer - Gene will be ON/OFF? Gene YPromoter

19. Positive Regulation & Constitutive Mutants Plus inducer - Gene will be ON/OFF? Gene YPromoter

20. Partial diploid containing a constitutive allele and a wild type allele has a constitutive phenotype THEREFORE, constitutive mutants are dominant/recessive to wild type under positive regulation Positive Regulation & Constitutive Mutants

21. Hypothesis araC is an activator of the L-ara operon Gene YPromoter araC - No L-ara OFF Gene YPromoter araC - Plus L-ara OFF Predictions araC c will be dominant/recessive to wild type araC Constitutive mutants, i.e. araC c, will be rare/common? araC- mutants cannot be induced with L-ara

22. Bahavior of araC c mutants Conclusion: araC c mutations are dominant/recessive to wild type araC CcCc BAD OFF CcCc BAD ON No L-arabinose Plus L- arabinose C+C+ BA D F’F’ C+C+ BA D F’F’

23. If araC c mutations are recessive to wild type, is araC a repressor? If araC is a repressor, why are araC c mutations supersuppressive and not constitutive? If araC is a repressor, why are araC c mutations rare?

24. AraC a repressor & an activator Revised Hypothesis: AraC is a repressor in the absence of L-ara but an activator in the presence of L-ara Gene YPromoter Wild type No L-ara OFF AraC Wild Type Plus L-ara Gene YPromoter RNAPol ON AraC

25. AraC a repressor & an activator BUT … why is araC c recessive to wild type? CcCc BAD OFF No L- arabinose AraC AraC c C+C+ BA D F’F’

26. AraC a repressor & an activator At least two models (not mutually exclusive) –AraC in its repressor conformation binds to DNA with higher affinity than AraC c mutants –AraC peptides form a complex (e.g. a homodimer) to be functional Gene YPromoter OFF AraC Gene YPromoter RNAPol ON AraC

27. Fig 12.20