Dissertation

Studies of the role of the transcriptional activator STE12 in the yeast pheromone response pathway

Yuan, Yi-lu Olive

1992

Overview

The STE12 protein of the yeast Saccharomyces cerevisiae binds to the pheromone response element (PRE) present in the upstream region of genes whose transcription is induced by pheromone. Using DNaseI footprinting assays with bacterially made STE12 fragments, I localized the DNA-binding domain to 164 amino acids near the amino terminus, a domain that contains similarities to the homeodomain. A DNaseI footprinting assay showed that the N-terminal 215 amino acids of STE12 has 5-10 fold higher binding affinity to two adjacent sites than to a single site. On a fragment containing multiple PREs, the 215 amino acid STE12 fragment protected both a consensus PRE as well as a degenerate PRE containing an additional residue. Mutation of the degenerate site led to a 5-10 fold decrease in binding; mutation of the consensus site led to a 25 fold decrease in binding. The ability of PREs to function as pheromone-inducible upstream activation sequences in yeast correlated with their ability to bind the STE12 domain in vitro. To investigate the role of STE12 in$\\alpha$ -specific gene activation, I cloned the STE12 functional homolog from the closely related yeast species Kluyveromyces lactis. The K. lactis STE12 (Kl STE12) contains 666 amino acids and is therefore similar in size to the S. cerevisiae STE12 (Sc STE12). The DNA-binding domains of these two STE12 proteins are highly conserved (78% identity) and they bind to the pheromone response element with similar affinity in vitro. However, the remainder of the proteins, required for uninduced transcription and pheromone-induced transcription, is very divergent (10 to 20% identity) with the exception of three short stretches of 9-15 residues. I introduced genes encoding Kl STE12 or both Kl STE12 and the K. lactis MAT $\\alpha$ 1 protein (Kl$\\alpha$ 1) into ste12 $\\Delta$strains of S. cerevisiae and examined their effects on mating. The Kl STE12 gene in low copy number can partially restore mating to S. cerevisiae a ste12 $\\Delta$cells, but little complementation occurs in$\\alpha$ste12$\\Delta$cells. However, the low level of Kl STE12 combined with Kl$\\alpha$ 1 complements the mating defect in$\\alpha$ste12 $\\Delta$cells. This enhancement suggests that STE12 may interact with$\\alpha$ 1 in a complex that is responsible for$\\alpha$ -specific gene activation. In addition, by generating hybrids of the S. cerevisiae and K. lactis STE12 proteins, I determined that the C-terminal activation domain of Kl STE12 is responsible for the inefficient mating observed in$\\alpha$ste12 $\\Delta$cells.

• Publisher: ProQuest Dissertations & Theses
• Subject: Genetics / Microbiology / Molecular biology
• ISBN: 9798209248415