The Transcriptional Activator GCN4 Contains Multiple Activation Domains That Are Critically Dependent on Hydrophobic Amino Acids

Abstract

GCN4 is a transcriptional activator in the bZIP family that regulates amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae. Previous work suggested that the principal activation domain of GCN4 is a highly acidic segment of approximately 40 amino acids located in the center of the protein. We conducted a mutational analysis of GCN4 with a single-copy allele expressed under the control of the native promoter and translational control elements. Our results indicate that GCN4 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 activation domain defined previously; the other extends over the N-terminal one-third of the protein. Both domains are partially dependent on the 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 critically dependent on bulky hydrophobic amino acids for their function. Five of the important hydrophobic residues, Phe-97, Phe-98, Met-107, Tyr-110, and Leu-113, fall within a region of proposed sequence homology between GCN4 and the herpesvirus acidic activator VP16. The remaining three residues, Trp-120, Leu-123, and Phe-124, are highly conserved 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 inactivate full-length GCN4. These hydrophobic residues may mediate important interactions between GCN4 and one or more of its target proteins in the transcription initiation complex.

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.…”

Membrane stress is coupled to a rapid translational control of gene expression in chlorpromazine-treated cells

“…The transcription of the HIS4 gene is known to be upregulated by Gcn4p under conditions of amino acid starvation (Drysdale et al 1995). We thus determined whether Gcn4p induced by CPZ increases the -galactosidase activity in cells containing the HIS4-lacZ reporter.…”

Modular organization of the E2F1 activation domain and its interaction with general transcription factors TBP and TFIIH

“…The analysis of particular transcriptional activators has sometimes revealed the presence of multiple activation domains within a single activator protein (Ma and Ptashne, 1987;Courey and Tjian, 1988;Goodrich et al, 1993;Tanaka and Herr, 1994;Drysdale et al, 1995). In certain of these cases, activation domains have been demonstrated to consist of minimal activation modules which, when reiterated, result in a synergistic activation of transcription (Tanaka and Herr, 1994;Blair et al, 1996), but the meaning of this observation has not been clear.…”