Analysis of the yeast Gal3 protein, a key component of the GAL gene transcription switch
Open Access
- Author:
- Diep, Cuong Quoc
- Graduate Program:
- Biochemistry and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 11, 2006
- Committee Members:
- James E Hopper, Committee Chair/Co-Chair
Anita Klein Hopper, Committee Member
Sergei A Grigoryev, Committee Member
George Makhatadze, Committee Member
Vincent Chau, Committee Member - Keywords:
- GAL gene switch
transcription regulation
GAL3 - Abstract:
- Transcriptional regulation of protein-encoding genes in eukaryotic organisms is required for proper development, differentiation, and response to environmental changes. The expression of specific genes at the transcriptional level is often tightly regulated to ensure that these genes are turned on or off only at the proper time and in the appropriate cell type. One of the most well studied cases of such regulation in eukaryotes is the GAL gene switch found in the yeast Saccharomyces cerevisiae. The GAL genes are necessary for the metabolism of galactose and their transcription is activated by a DNA-binding transcriptional activator, Gal4, in the presence of galactose. However, without galactose the activity of Gal4 is inhibited by its interaction with Gal80. A key regulatory component in activation of the GAL gene switch is the Gal3 protein. Gal3 senses the presence of intracellular galactose and binds to Gal80, relieving the Gal80-inhibition of Gal4. Although Gal3 interaction with Gal80 leads to Gal4-mediated activation of the GAL genes, there is a major gap of knowledge in how Gal3 itself is activated by galactose and ATP, and it is not known which amino acids of Gal3 are important for interaction with Gal80. In this thesis work, I addressed the mechanism in which Gal3 is activated by its ligands and also identified the surface and amino acids that are necessary for interaction with Gal80. Intragenic suppression analyses were performed for GAL3C mutants that confer a constitutive GAL gene activation and a galactose-independent interaction with Gal80. Of the five GAL3C alleles analyzed, four were suppressed by the second-site suppressor (GAL3SOC-D68S), while one allele (GAL3C-D368V) was not suppressed. On the Gal3 homology model, the suppressed GAL3C alleles co-localized with the second-site suppressor to the “hinge” region consisting of β-sheets s-C, -H and α-helices h-K, -O, whereas the non-suppressed allele localized to the opposite side of the “hinge” region at α-helix h-J. The GAL3C-D368V allele had previously been shown to suppress a GAL80S-G323R interaction mutation. Crosslinking results of Gal3 and Gal80 are consistent with the notion that they interact by surfaces that include Gal3-D368 and Gal80-G323. This is further supported by the localization of gal3 interaction mutants surrounding Gal3-D368 on the homology model. The data suggest that a local conformational change occurs within the “hinge” region to activate Gal3, while the interaction interface encompassing D368 plays a direct role in the binding to Gal80. Furthermore, based on considerations of E. coli galactokinase and previous attempts to isolate Gal3 interaction peptides, I conclude that the Gal80-binding surface of Gal3 is a composite of noncontiguous elements.