Structure/functional properties of the yeast dual regulator protein NGG1 that are required for glucose repression.

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).

Characterization of NGG1, a novel yeast gene required for glucose repression of GAL4p-regulated transcription.

The GAL1-10 genes of Saccharomyces cerevisiae are regulated by the interaction of cis- and trans-acting factors which facilitate activated transcription in galactose but not in glucose medium. By selecting mutations that allow expression of a defective gal1-10-his3 hybrid promoter, we have identified a novel gene, NGG1, which is required for glucose repression of the GAL10-related his3-G25 promoter. ngg1 was identified as a recessive null mutation that in the presence of a gal80 background resulted in a 300-fold relief of glucose repression for the his3-G25 promoter. This compared with a 20-fold and negligible relief of repression in gal80 and ngg1 strains, respectively. Deletion analysis of the his3-G25 promoter showed a correlation between the number of GAL4p binding sites and the relative level of NGG1p activity. Relief of glucose repression by NGG1 was dependent on the presence of GAL4, but was independent of the GAL4 promoter. In addition, NGG1p activity was seen for a promoter construct containing independent GAL4p binding sites. These results suggest that NGG1p acts to inhibit GAL4p function in glucose medium. We have cloned NGG1 by complementation and found that it contains an open reading frame of 2106 bp which could encode a protein with a molecular weight of 79,230.

TATA-binding protein activates transcription when upstream of a GCN4-binding site in a novel yeast promoter.

In the gal-his3 hybrid promoter, his3-GG1, GCN4 stimulates transcription at the position normally occupied by a TATA element. This expression requires two elements within gal1-10 sequences, a REB1-binding site and a second element, Z, which resides 20 base pairs upstream of the GCN4-binding site. No obvious TATA element is present in this promoter. To characterize the function of Z, we replaced it with short random oligonucleotides and selected for expression in vivo. Fourteen elements were identified and classified into groups based upon sequence and phenotypic similarities. Group 1 elements contained functional TATA sequences that were essential for activity. TATA elements can thus function when positioned upstream of a GCN4-binding site. The Group 2 elements activated transcription poorly when used as conventional TATA elements; however, mutational analyses demonstrated that their activity required TATA-like sequences. These TATA-like sequences bound the yeast TATA-binding protein (TBP) poorly in vitro but function in vivo as TBP interaction sites based upon two criteria. First mutations that improved their TATA character correspondingly improved function and second their activity could be enhanced in the presence of an altered binding specificity mutant of TBP. Furthermore, the Group 2 elements enabled the identification of mutations outside of the TATA-like core that contribute to transcriptional activation without adversely affecting TBP binding. The finding that low affinity TBP-binding sites can be used at unconventional positions suggests that many "TATA-less" promoters contain a cryptic interaction site for TBP.