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 MCB 140 11 20 06 1 Simple experiment 1985 GAL1 promoter fragment HIS3 promoter HIS3 glucose Transcription of HIS3 gene is repressed even under conditions when the HIS3 promoter is maximally active amino acid starvation Struhl K Nature 317 822 1985 MCB 140 11 20 06 2 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 MCB 140 11 20 06 3 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 MCB 140 11 20 06 4 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 MCB 140 11 20 06 5 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 MCB 140 11 20 06 6 Bill Watterson MCB 140 11 20 06 7 1986 SNF1 is a protein kinase Glucose present Snf1p inactive Glucose absent Snf1p active phosphorylates something and that something stops repressing the sugarutilizing gene promoters The search for something DOWNSTREAM of SNF1 Celenza JL Carlson M Science 233 1175 1986 MCB 140 11 20 06 8 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 Sch ller HJ Entian KD J Bacteriol 173 2045 1991 MCB 140 11 20 06 9 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 MCB 140 11 20 06 10 MCB 140 11 20 06 11 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 MCB 140 11 20 06 12 Life or death GAL1 promoter death galactose cell rapidly dies MCB 140 11 20 06 13 Mass action in action Overexpress something that will compete with Gal4p activating the promoter MCB 140 11 20 06 14 What part II found MIG1 multicopy inhibitor of GAL genes MCB 140 11 20 06 15 CAT4 epistatic to SNF1 MIG1 silences GAL1 promoter MCB 140 11 20 06 16 They are the same gene CAT4 MIG1 Mig1p DNA binding protein transcriptional repressor MCB 140 11 20 06 17 MCB 140 11 20 06 18 MCB 140 11 20 06 19 How genes respond to environmental stimuli MCB 140 11 20 06 20 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 MCB 140 11 20 06 21
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