IDENTIFICATION OF 7 HYDROPHOBIC CLUSTERS IN GCN4 MAKING REDUNDANT CONTRIBUTIONS TO TRANSCRIPTIONAL ACTIVATION

GCN4 is a transcriptional activator in the bZIP family that regulates amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae. T he N-terminal 100 amino acids of GCN4 contains a potent activation fun ction that confers high-level transcription in the absence of the cent rally located acidic activation domain (CAAD) delineated in previous s tudies, To identify specific amino acids important for activation by t he N-terminal domain, we mutagenized a GCN4 allele lacking the CAAD an d screened alleles in vivo for reduced expression of the HIS3 gene, We found four pairs of closely spaced phenylalanines and a leucine resid ue: distributed throughout the N-terminal 100 residues of GCN4 that ar e required for high-level activation in tile absence of the CAAD. Trp, Leu, and Tyr were highly functional substitutions for the Phe residue at position 45. Combined with our previous findings, these results in dicate that GCN4 contains seven clusters of aromatic or bulky hydropho bic residues which make important contributions to transcriptional act ivation at HIS3. None of the seven hydrophobic clusters is essential f or activation by full-length GCN4, and the critical residues in two or three clusters must be mutated simultaneously to observe a substantia l reduction in GCN4 function, Numerous combinations of four or five in tact clusters conferred high-level transcription of HIS3. We propose t hat many of the hydrophobic clusters in GCN4 act independently of one another to provide redundant means of stimulating transcription and th at the functional contributions of these different segments are cumula tive at the HIS3 promoter, On the basis of the primacy of bulky hydrop hobic residues throughout the activation domain, we suggest that GCN4 contains multiple sites that mediate hydrophobic contacts with one or more components of the transcription initiation machinery.