1. MCB 140 11-20-06 1 Further conservation Induction of GAL genes by galactose is only half the story. The GAL genes are also severely repressed by glucose (the same is true of the lac genes in E. coli). In fact, all non-glucose sugar utilization circuits in yeast are repressed by glucose.

2. MCB 140 11-20-06 2 Simple experiment (1985) GAL1 promoter fragment HIS3 promoter HIS3 Struhl K. Nature 317: 822 (1985). + glucose Transcription of HIS3 gene is repressed, even under conditions when the HIS3 promoter is maximally active! (amino acid starvation)

3. MCB 140 11-20-06 3 Conclusion Normally, his3 expression … occurs at a basal level which can be induced by conditions of amino-acid starvation. However, in glucose medium, the catabolite regulatory sequence overrides the normal his3 promoter elements and reduces transcription both in normal and starvation conditions. The implication from these results is that in contrast to catabolite repression in Escherichia coli, which is mediated by catabolite-activating protein (CAP), catabolite repression in yeast occurs by a negative control mechanism involving a putative repressor protein. The observation that this regulatory site exerts its repressing effects even when located upstream of an intact promoter region suggests that repression in yeast is not mediated by steric interference between regulatory proteins and the transcriptional apparatus. Struhl K. Nature 317: 822 (1985).

4. MCB 140 11-20-06 4 This is what this means There must be some REPRESSOR that is glucose-responsive – it kills sugar-utilizing enzyme gene promoters when glucose is present. Under low glucose, this repressor goes away. How to find that repressor?

5. MCB 140 11-20-06 5 Death by sugar Marian Carlson screened for mutants that would be sucrose non-fermenters (snf) – that is, would not grow on sucrose in the absence of glucose. 1.Enzymes that metabolize sucrose. 2.“Suc4” – the positive regulator that responds to sucrose addition (no such thing). 3.The “glucose sensor” – if you mutate it, the cell will think glucose is present! Carlson M. et al. Genetics 98: 25 (1981).

6. MCB 140 11-20-06 6 What M. Carlson found 1.suc2 – mutations in invertase, the key enzyme that breaks down sugar. 2.snf1 – an unlinked mutation. “The snf1 mutations were found to be pleiotropic, preventing sucrose utilization by SUC2+ and SUC7+ strains, and also preventing utilization of galactose, maltose and several nonfermentable carbon sources. “ Carlson M. et al. Genetics 98: 25 (1981).

7. MCB 140 11-20-06 7  Bill Watterson

8. MCB 140 11-20-06 8 1986: SNF1 is a protein kinase Glucose present = Snf1p inactive Glucose absent = Snf1p active, phosphorylates something, and that something stops repressing the sugar- utilizing gene promoters. The search for something DOWNSTREAM of SNF1. Celenza JL, Carlson M. Science 233: 1175 (1986).

9. MCB 140 11-20-06 9 Search for something, part I Classical epistasis screen – search for mutations that would be epistatic to SNF1. 1.Take snf1 cells (do not grow on sucrose). 2.Mutagenize the poor things. 3.Cells that grow on sucrose contain an extragenic suppressor of the snf1 mutation. Schüller HJ, Entian KD. J. Bacteriol. 173: 2045 (1991).

10. MCB 140 11-20-06 10 What was found in part I New gene, CAT4. Mutations in CAT4 nicely suppressed the growth defect of snf1 cells. In other words, snf1 cat4 cells do not exhibit a sucrose nonfermentation phenotype. = Cat4p acts downstream of Snf1p.

11. MCB 140 11-20-06 11

12. MCB 140 11-20-06 12 Search for something, part II Find a protein the overexpression of which will shut down the GAL1 promoter. Even in the presence of galactose. High-copy suppressor screen: 1. Take mutant cell. 2. Transform a library of cDNAs driven by a strong promoter. 3. Find cDNA that suppresses mutant phenotype. Nehlin JO, Ronne H. EMBO J. 9: 2891 (1990).

13. MCB 140 11-20-06 13 Life or death GAL1 promoter death + galactose cell rapidly dies

14. MCB 140 11-20-06 14 Mass action in action Overexpress something that will compete with Gal4p activating the promoter:

15. MCB 140 11-20-06 15 What part II found MIG1 (multicopy inhibitor of GAL genes).

16. MCB 140 11-20-06 16 CAT4 – epistatic to SNF1. MIG1 – silences GAL1 promoter.

17. MCB 140 11-20-06 17 They are the same gene. CAT4 = MIG1 Mig1p – DNA-binding protein (transcriptional repressor)

18. MCB 140 11-20-06 18

19. MCB 140 11-20-06 19

20. MCB 140 11-20-06 20 How genes respond to environmental stimuli

21. MCB 140 11-20-06 21 More from Dr. Jacob “I have always been convinced that the same principles operating in bacteria are also operating in higher organisms with added complexity. The question therefore is to understand what kind of complexity is involved and how it is generated.”