Identification of Seven Hydrophobic Clusters in GCN4 Making Redundant Contributions to Transcriptional Activation

Abstract

GCN4 is a transcriptional activator in the bZIP family that regulates amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae. The N-terminal 100 amino acids of GCN4 contains a potent activation function that confers high-level transcription in the absence of the centrally located acidic activation domain (CAAD) delineated in previous studies. To identify specific amino acids important for activation by the N-terminal domain, we mutagenized a GCN4 allele lacking the CAAD and screened alleles in vivo for reduced expression of the HIS3 gene. We found four pairs of closely spaced phenylalanines and a leucine residue distributed throughout the N-terminal 100 residues of GCN4 that are required for high-level activation in the 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 indicate that GCN4 contains seven clusters of aromatic or bulky hydrophobic residues which make important contributions to transcriptional activation at HIS3. None of the seven hydrophobic clusters is essential for activation by full-length GCN4, and the critical residues in two or three clusters must be mutated simultaneously to observe a substantial reduction in GCN4 function. Numerous combinations of four or five intact clusters conferred high-level transcription of HIS3. We propose that many of the hydrophobic clusters in GCN4 act independently of one another to provide redundant means of stimulating transcription and that the functional contributions of these different segments are cumulative at the HIS3 promoter. On the basis of the primacy of bulky hydrophobic 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.

Activator Gcn4 Employs Multiple Segments of Med15/Gal11, Including the KIX Domain, to Recruit Mediator to Target Genes in Vivo

“…Thus, although Gcn4 appears to make multiple, low affinity interactions with several Gal11 regions, Pdr1 and Oaf1 interact more tightly, and perhaps exclusively, with the KIX domain. This view is consistent with our previous finding that the Gcn4 activation domain has a random coil structure in solution (63) and contains seven different clusters of hydrophobic residues scattered throughout the acidic activation domain, whose functions in Gcn4-Mediator interactions and transcriptional activation are highly redundant (47,61,62).…”

“…Supporting the conclusion that Gal11 contains independently functioning interaction modules, we found that distinct recombinant Gal11 polypeptides containing the KIX domain, a region altered by ⌬5 and the WQV substitution, and a region encompassing ⌬8 all bind to recombinant Gcn4 in vitro in a manner enhanced by the hydrophobic residues in Gcn4 necessary for Mediator binding to Gcn4 in vitro and transcriptional activation by Gcn4 in vivo (47,61,62). Furthermore, we used NMR chemical shift mapping to provide evidence that the recombinant Gcn4 activation domain interacts specifically with the KIX domain in solution, contacting a surface that overlaps with, but is distinct from, surfaces contacted by segments of other yeast or mammalian activation domains.…”

Artificial oncoproteins: modified versions of the yeast bZip protein GCN4 induce cellular transformation

“…In yeast, GCN4 and VP16 require different mediator subunits for activated transcription of specific genes (Myers et al, 1999). This suggests that the TADs of GCN4 and VP16, both of which are acidic, may operate by different mechanisms and interact with overlapping but nonidentical spectra of downstream targets (Jackson et al, 1996). The TADs of GCN4 and of VP16 also interact with the chromatin remodeling complex SWI/SNF (Liberati et al, 1999).…”

Recruitment of the ArgR/Mcm1p repressor is stimulated by the activator Gcn4p: A self-checking activation mechanism

“…The ability of Gcn4p to interact with coactivators in cell extracts (16,17) and to recruit coactivators to target promoters in vivo (14,18) depends on the hydrophobic clusters in its activation domain required for transcriptional activation in vivo (19). To determine whether recruitment of ArgR͞Mcm1p by Gcn4p involves its activation domain, we conducted ChIP assays on isogenic strains expressing wild-type Gcn4p or a mutant harboring Ala substitutions in all seven hydrophobic clusters in the activation domain (gcn4-14Ala).…”