The Interaction between an Acidic Transcriptional Activator and Its Inhibitor

Abstract

The GAL genes, which encode the enzymes required for normal galactose metabolism in yeast, are transcriptionally regulated by three proteins: Gal4p, an activator; Gal80p, an inhibitor; and Gal3p, a galactose sensor. These proteins control the switch between inert and active gene expression. The transcriptional activation function of Gal4p is rendered inactive in the presence of Gal80p. Here we present the three-dimensional structure of a complex between the acidic activation domain of Gal4p and Gal80p. The transactivation domain initiates with an extended region of polypeptide chain followed by two turns of an amphipathic ␣-helix. It fits into and across a deep cleft within the Gal80p dimer with the protein-protein interface defined primarily by hydrophobic interactions. A disordered loop in the apo-Gal80p structure (Asp-309 to Ser-316) becomes well-defined upon binding of the transactivation domain. This investigation provides a new molecular scaffold for understanding previous biochemical and genetic studies.In yeast, the GAL genes encode the enzymes of the Leloir pathway, which are required for the conversion of galactose into a metabolically useful form, glucose 6-phosphate (1). The regulation of these genes in response to the organism being challenged with galactose has served as a research paradigm for eukaryotic transcriptional control for over 50 years (1-5). Three key protein components form the GAL regulatory switch: a transcriptional activator, Gal4p; a transcriptional inhibitor, Gal80p; and an inducer, Gal3p. When yeast cells are grown in the absence of galactose, the GAL genes are, for the most part, transcriptionally inert. Under these conditions, Gal4p is produced in the cell and is tethered upstream of the GAL genes (6), but its activity is inhibited by its interaction with Gal80p (7). When galactose is available as a carbon source, the GAL genes are transcribed, both rapidly and to a high level (8). Although the presence of galactose within the cell triggers the activation of Gal4p, neither Gal4p nor Gal80p function as the galactose sensor. Instead, Gal3p serves in this capacity by binding both galactose and ATP and adopting the conformation required for its interaction with Gal80p (1, 9). The net result of this interaction is that Gal4p becomes active, and transcription of the GAL genes proceeds.Gal4p is a large protein of 881 amino acids with the first ϳ100 residues functioning in DNA recognition and dimerization. The last C-terminal residues serve as an acidic transactivation domain (TAD) 3 that is required, ultimately, for the recruitment of RNA polymerase II to initiate transcription (10). The amino acid residues comprising the TAD also provide the binding platform for Gal80p (11,12). Both Gal3p and Gal80p are smaller proteins containing ϳ520 and ϳ450 amino acids, respectively.Until very recently, the only three-dimensional structural information available for any of these key regulatory components of the GAL genetic switch was that of an N-terminal 65-residue fragment of Gal4p bound to D...

The Gal3p transducer of the GAL regulon interacts with the Gal80p repressor in its ligand-induced closed conformation

“…Comparison of the Gal3p-Gal80p-galactose-ATP structure presented here with the Gal80p-Gal4pAD structures from S. cerevisiae that we determined previously (Kumar et al 2008) and with the Gal80p-Gal4pAD structure from K. lactis (Thoden et al 2008) shows that Gal3p binds to a different surface on Gal80p than Gal4p-AD (Fig. 5).…”

“…However, this would not appear to change the conformation of Gal80p enough to prevent interaction with the Gal4p-TAD. That said, we only have a very small portion of Gal4p in the two crystal structures available (Kumar et al 2008;Thoden et al 2008). A tripartite complex between the three proteins can be formed in vitro (Platt and Reece 1998), albeit with a constitutive mutant of Gal3p and a much-truncated form of Gal4p.…”

“…This loop has a high B factor (average of 95.5 Å 2 ) in the Gal80p S2 structure and is disordered in the Gal80p S0 -Gal4AD-NAD complex. However, the corresponding loop in K. lactis Gal80p does become ordered upon Gal4p-TAD binding (Thoden et al 2008).…”

Interplay of a Ligand Sensor and an Enzyme in Controlling Expression of the Saccharomyces cerevisiae GAL Genes

“…To date, high-resolution three-dimensional structures are available for Gal80p (26) and for complexes between Gal80p and peptides representing the activation domain of Gal4p (11,25). In addition, the structure of Gal1p has been solved (27).…”

An N-terminal region of a Myb-like protein is involved in its intracellular localization and activation of a gibberellin-inducible proteinase gene in germinated rice seeds

“…The GAL4 activation domain, which is also rich in acidic amino acids, interacts with a repressor. 34) Therefore, the amino acids in the N-terminal domain of CTMyb1L may be involved in the determination of CTMyb1L localization by interacting with unknown proteins in this domain.…”