Poster abstracts

Poster number 27 submitted by Shuo Jin

Isolation and characterization of Gal80 self-association defective mutants

Shuo Jin (MCDB program and The Ohio State University), Sudip Goswami (Molecular Genetics and The Ohio State University)

Abstract:
Abstract:
Cells have developed genetic switches that control the activation and repression of transcription of specific groups of genes in response to environmental changes. One such genetic switch is the GAL gene switch that controls the yeast galactose pathway genes (GAL genes). The GAL genes are induced by galactose through the activities of three regulatory proteins, Gal4, Gal80, and Gal3. Gal4, the transcriptional activator, binds as a dimer to the promoter of GAL genes both in the presence and absence of galactose. In the absence of galactose, Gal80 binds to and inhibits the Gal4 transcription activation domain (AD). In the presence of galactose, Gal3 binds to Gal80, relieving Gal80 inhibition of Gal4. Pilauri et al. found that Gal80 variants selected for impaired binding to Gal4AD retained their capacities to bind to Gal3 and to self-associate, whereas those selected for impaired binding to Gal3 lost their capacities to bind to Gal4AD and self-associate. Based on those results they proposed that Gal3 binding to a monomer of Gal80 competes with Gal80-Gal80 self-association thereby reducing the availability of dimer, the form postulated to be required for binding to Gal4. However, there has been no study to determine whether Gal80 mutations that impair Gal80 self-association also impair binding to Gal4AD or Gal3. Here we address this by directly selecting for Gal80 variants that fail to self-associate and testing them for ability to associate with Gal4 and Gal3. By PCR-mutagenesis and yeast reverse 2-hybrid selections we isolated six Gal80 mutants with varying degrees of self-association impairment. All six failed to interact with Gal4AD. Three of the six failed to interact with Gal3, whereas the other three with relatively less severe deficiency in self-association showed some interaction with Gal3. These results are consistent with both the model proposed by Pilauri et al. and a mechanism wherein Gal3 binding to the dimer of Gal80 triggers dissociation to monomers.

References:
Pilauri V, Bewley M, Diep C,Hopper J (2005) Gal80 dimerization and the yeast GAL gene switch. Genetics 169: 1903-1914

Keywords: Gal80, Gal4AD, Gal3