Regulated expression of the GAL4 activator gene in yeast provides a sensitive genetic switch for glucose repression.

Abstract

Glucose (catabolite) repression is mediated by multiple mechanisms that combine to regulate transcription of the GAL genes over at least a thousandfold range. We have determined that this is due predominantly to modest glucose repression (4-to 7-fold) of expression of GAL4, the gene encoding the transcriptional activator of the GAL genes. GALA regulation is affected by mutations in several genes previously implicated in the glucose repression pathway; it is not dependent on GAL4 or GAL80 protein function. GALA promoter sequences that mediate glucose repression were found to lie downstream of positively acting elements that may be "TATA boxes." Two nearly identical sequences (10/12 base pairs) in this region that may be binding sites for the MIG1 protein were identified as functional glucose-control elements. A 4-base-pair insertion in one of these sites causes constitutive GAL4 synthesis and leads to substantial relief (50-fold) of glucose repression ofGAL] expression. Furthermore, promoter deletions that modestly reduce GALA expression, and therefore presumably the amount of GAL4 protein synthesized, cause much greater reductions in GAL] expression. These results suggest that GALA works synergistically to activate GALl expression. Thus, glucose repression of GAL) expression is due largely to a relatively small reduction of GALA protein levels caused by reduced GALA transcription. This illustrates how modest regulation of a weakly expressed regulatory gene can act as a sensitive genetic switch to produce greatly amplifiled responses to environmental changes.Expression of the GAL], -7, and -10 genes, which are required for galactose catabolism in Saccharomyces cerevisiae, is regulated at two levels (1). (i) Galactose induces their transcription by preventing GAL80 protein from inhibiting function of the GAL4 transcriptional activator. (ii) Glucose causes severe repression of GAL gene transcription by a process to which several different mechanisms contribute. Some operate to reduce the amount of inducer available to inactivate GAL80 by reducing expression of GAL3, required for inducer synthesis (2), and of GAL2, encoding the galactose transporter (3), and by inactivation of preexisting galactose permease in the cell (4). Other mechanisms of repression operate through sites in the GAL promoters termed the upstream activation sequence (UAS) and the upstream repression sequence (URS) and, therefore, act more directly to repress transcription.The UAS and URS regions from the GAL] promoter are capable of independently mediating glucose repression (5). The repression that operates through the UAS region, which contains four binding sites for the GAL4 activator, probably reflects reduced levels or reduced function of the GAL4 protein in glucose-grown cells. Repression mediated by the URS, which lies between the UAS and the "TATA element," is presumably due to unidentified repressors that bind to this region.UAS-mediated repression is characterized by the failure of GAL4 to bind the UAS in cells growing in the pr...

A family of hexosephosphate mutases in Saccharomyces cerevisiae

“…Fragments of genomic DNA with sizes between 3.0-3.4 kb and 4.3-4.7 kb, respectively, corresponding to the size of the expected PGM2 gene (Oh and Hopper, 1990) were cloned into plasmid YEplacl81. Both gene li- (Struhl, 1986), transcription-termination signals (Zaret and Sherman, 1982), and putative binding sites for the GAL4 activator (West et al, 1984;Tajima et al, 1986) and MIGl repressor proteins (Nehlin et al, 1991;Griggs and Johnston, 1991) are printed in bold and are underlined. The active-site serine residues are marked by asterisks.…”

Regulated nuclear translocation of the Mig1 glucose repressor.

“…Preference for glucose is in part due to repression of transcription of genes not required for growth on glucose, such as genes encoding enzymes for converting other carbon sources to glycolytic intermediates (e.g., GAL and SUC), gluconeogenesis (e.g., FBP1 and PCK1), and respiration (e.g., CYCI and COX6) (for reviews, see Johnston and Carlson, 1992;Trumbly, 1992;Ronne, 1995). Migl is the repressor responsible for glucose repression of many genes, including GAL, SUC, and MAL (Nehlin and Ronne, 1990;Griggs and Johnston, 1991;Nehlin et al, 1991;Schuller and Entian, 1991;Flick and Johnston, 1992;Johnston et al, 1994;Vallier and Carlson, 1994;Hu et al, 1995;Lutfiyya and Johnston, 1996;Wang and Needleman, 1996). Migl is a Cys2-His2 zinc finger-containing protein that binds to promoters of glucose-repressed genes and recruits the general repressors Ssn6 and Tupl Struhl, 1994, 1995;Treitel and Carlson, 1995).…”

MIG1‐dependent and MIG1‐independent regulation of GAL gene expression in Saccharomyces cerevisiae: role of Imp2p

“…Since the level of GAL4 expression is rate-limiting for the induction of GAL gene expression (Griggs and Johnston, 1991) and the expression of GAL4 itself is under the control of IMP2, we tested whether imp2 glucose repressible phenotype was influenced by GAL4 gene dosage.…”

“…Gal4p is in turn glucose-regulated (Laughon and Gesteland, 1982;Griggs and Johnston, 1991;Nehlin et al, 1991). It has been demonstrated that modest glucose repression (four-to seven-fold) of GAL4 is the predominant cause of glucose repression of GAL genes (Griggs and Johnston, 1991). Mig1p is mainly responsible for glucose repression of GAL genes (Nehlin et al, 1991;Flick and Johnston, 1992;Johnston et al, 1994).…”

“…Galactose induction is mediated by Gal4p, which transcriptionally activates the genes involved in galactose catabolism, binding to their promoters. Gal4p is in turn glucose-regulated (Laughon and Gesteland, 1982;Griggs and Johnston, 1991;Nehlin et al, 1991). It has been demonstrated that modest glucose repression (four-to seven-fold) of GAL4 is the predominant cause of glucose repression of GAL genes (Griggs and Johnston, 1991).…”