Abstract
NGG1p/ADA3p is a yeast dual function regulator required for the complete glucose repression of GAL4p-activated genes (Brandl, C. J., Furlanetto, A. M., Martens, J. A., and Hamilton, K. S. (1993) EMBO J. 12, 5255-5265). Evidence for a direct role for NGG1p in regulating activator function is supported by the finding that NGG1p is also required for transcriptional activation by GAL4p-VPl6 and LexA-GCN4p (Pina, B., Berger, S. L., Marcus, G. A., Silverman, N., Agapite, J., and Guarente, L. (1993) Mol. Cell. Biol. 13, 5981-5989). By analyzing deletion derivatives of the 702-amino acid protein, we identified a region essential for glucose repression within residues 274-373. Essential sequences were further localized to a segment rich in Phe residues that is predicted to be an amphipathic alpha helix. As well as finding mutations within this region that reduced glucose repression, we identified mutations that made NGG1p a better repressor. In addition, NGG1p probably represses GAL4p activity as part of a complex containing ADA2p because single and double disruptions of ngg1 and ada2 had comparable effects on glucose repression. We also localized a transcriptional activation domain within the amino-terminal amino acids of NGG1p that is proximal or overlapping the region required for glucose repression. Activation by GAL4p-NGG1p(1-373) requires ADA2p; however, activation by GAL4p-NGG1p(1-308), is ADA2p-independent. This suggests that a site required for ADA2p interaction lies between amino acids 308 and 373 and that ADA2p has a regulatory role in activation by GAL4p-NGG1p(1-373).
Highlights
The genes required for galactose metabolism in Saccharomyces cerevisiae provide one of the principal model systems for the interfacing between positive and negative transcriptional regulatory networks
Regions of NGG1p Required for Glucose Repression—To initiate studies into the regions of NGG1p required for glucose repression, we analyzed deletion derivatives of the gene for their ability to repress expression of GAL10 in glucose medium
In this experiment disruption of NGG1 resulted in a 7.7-fold decrease in glucose repression in comparison with a wild type NGG1 allele expressed from the ded1 promoter
Summary
The genes required for galactose metabolism in Saccharomyces cerevisiae provide one of the principal model systems for the interfacing between positive and negative transcriptional regulatory networks. These genes are induced approximately 1000-fold in a process that requires the GAL4p transcriptional activator protein (reviewed in Ref. 1). Binding of MIG1p [15] to the GAL4 promoter results in decreased transcription of GAL4 of approximately 5-fold in glucose-containing medium [16, 17]. Direct action of NGG1p on the function of transcriptional activators was suggested by the finding that nonfunctional mutations of ngg suppress the lethal effects of overexpression of GAL4pVP16 fusions [29]. This suppression was due to reduced activation by GAL4p-VP16 in this background. Pina et al [27] and Georgakopoulos et al [30] demonstrated that NGG1p is required for maximal transcriptional activation by a group of activators that includes GAL4p-VP16 and LexA-GCN4
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