UBC Theses and Dissertations
Structure-function analysis of the MAT[Alpha]2 protein of Saccharomyces cerevisiae Ho, Chi-Yip
In Saccharomyces cerevisiae, the MATα2 gene encodes a negative regulatory protein responsible for determining cell-specific mating-type. The MATα2 protein, α2, represses two sets of mating-type-specific genes by interacting with two other factors. α2 forms a heterodimer with the repressor protein a1, and binds a DNA-target site located upstream of all haploid-specific genes, thereby repressing these genes. In combination with a second factor, MCM1, α2 recognizes another DNA-target site found in all the a-specific genes. The binding of α2-MCM1 to these sites leads to the repression of a-specific genes. Therefore, α2 carries out a dual regulatory function in mating-type control through protein-protein interactions with its co- repressors and through the changes in DNA-binding specificities upon interactions with these partners. The objective of this thesis is to gain further insight (i) into the heterodimerization of α2 with al and (ii) into DNA-binding specificity of α2 when acting with MCMI. In studies to localize the important structures within α2 for functional al-α2 heterodimerization, two sequences in the N-terminal half of α2, containing the 3,4-hydrophobic heptad repeat pattern characteristic of coiled-coils, were identified and examined. Amino acid replacements showed that particular positions within the heptads are important in a1-α2 repression and in stabilizing the a1-α2 heterodimer. These mutational analyses are consistent with the proposal that the two heptad sequences of α2 associate with a1 in a coiled-coil-like manner. To determine the DNA-binding specificity of α2 in the presence of MCM1, a variant of the wild-type α2 operator was used as the DNA-target in an in vitro electrophoretic band-shift assay. This variant α2 operator was also used in a heterologous reporter gene system to test the in vivo repression mediated by α2-MCM1. The results together indicate that the nucleotide differences in the variant operator caused a decrease in both α2-MCM1 binding and repression. This suggests that nucleotides outside the core consensus sequence of the α2 operator are important for its in vivo recognition by α2-MCM1.
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