THE TRANSCRIPTIONAL ACTIVATOR GCN4 CONTAINS MULTIPLE ACTIVATION DOMAINS THAT ARE CRITICALLY DEPENDENT ON HYDROPHOBIC AMINO-ACIDS

GCN4 is a transcriptional activator in the bZIP family that regulates amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae, P revious work suggested that the principal activation domain of GCN4 is a highly acidic segment of approximately 40 amino acids located in th e center of the protein, We conducted a mutational analysis of GCN4 wi th a single-copy allele expressed under the control of the native prom oter and translational control elements, Our results indicate that GCN 4 contains two activation domains of similar potency that can function independently to promote high-level transcription of the target genes HIS3 and HIS4. One of these domains is coincident with the acidic act ivation domain defined previously; the other extends over the N-termin al one-third of the protein, Both domains are partially dependent on t he coactivator protein ADA2. Each domain appears to be composed of two or more small subdomains that have additive effects on transcription and that can cooperate in different combinations to promote high-level expression of HIS3 and HIS4. At least three of these subdomains are c ritically dependent on bulky hydrophobic amino acids for their functio n, Five of the important hydrophobic residues, Phe-97, Phe-98, Met-107 , Tyr-110, and Leu-113, fall within a region of proposed sequence homo logy between GCN4 and the herpesvirus acidic activator VP16. The remai ning three residues, Trp-120, Leu-123, and Phe-124, are highly conserv ed between GCN4 and its Neurospora counterpart, cpc-1. Because of the functional redundancy in the activation domain, mutations at positions 97 and 98 must be combined with mutations at positions 120 to 124 to observe a substantial reduction in activation by full-length GCN4, and substitution of all eight hydrophobic residues was required to inacti vate full-length GCN4, These hydrophobic residues may mediate importan t interactions between GCN4 and one or more of its target proteins in the transcription initiation complex.