TRANSCRIPTIONAL ACTIVATION UPON PHEROMONE STIMULATION MEDIATED BY A SMALL DOMAIN OF SACCHAROMYCES-CEREVISIAE STE12P

In the yeast Saccharomyces cerevisiae, Ste12p induces transcription of pheromone-responsive genes by binding to a DNA sequence designated th e pheromone response element. We generated a series of hybrid proteins of Ste12p with the DNA-binding and activation domains of the transcri ptional activator Gal4p to define a pheromone induction domain of Ste1 2p sufficient to mediate pheromone-induced transcription by these hybr id proteins. A minimal pheromone induction domain, delineated as resid ues 301 to 335 of Ste12p, is dependent an the pheromone mitogen-activa ted protein (MAP) kinase pathway for induction activity. Mutation of t he three serine and threonine residues within the minimal pheromone in duction domain did not affect transcriptional induction, indicating th at the activity of this domain is not directly regulated by MAP kinase phosphorylation. By contrast, mutation of the two tyrosines or their preceding acidic residues led to a high level of transcriptional activ ity in the absence of pheromone and consequently to the loss of pherom one induction. This constitutively high activity was not affected by m utations inn the MAP kinase cascade, suggesting that the function of t he pheromone induction domain is normally repressed in the absence of pheromone. By two-hybrid analysis, this minimal domain interacts with two negative regulators, Dig1p and Dig2p (also designated Rst1p and Rs t2p), and the interaction is abolished by mutation of the tyrosines. T he pheromone induction domain itself has weak and inducible transcript ional activity, and its ability to potentiate transcription depends on the activity of an adjacent activation domain. These results suggest that the pheromone induction domain of Ste12p mediates transcriptional induction via a two-step process: the relief of repression and synerg istic transcriptional activation with another activation domain.