Master of Science
Dr. Chris Brandl
The regulation of transcription is an important cellular function because it is the first step in gene regulation. In Saccharomyces cerevisiae, two protein complexes, SAGA and NuA4, act as regulators of transcription. A common protein shared between these two complexes, called Tra1, regulates transcriptional activation through its interaction with gene specific transcriptional activators. Tra1 is a member of the PIKK family of proteins, which are characterized by FAT, PI3K and FATC domains. The FATC domain encompasses the terminal 33-35 residues of the protein. Two mutations within the FATC domain, tra1-L3733A and tra1-F3744A, result in slow growth under stress conditions. Partially dominant mutations in the gene encoding Tti2, a 421 residues component of the TTT chaperone complex, suppress these phenotypes. My goal was to further characterize the role of the FATC domain of Tra1 by determining which residues are important for function, and characterize how these relate to the TRA1-TTI2 interaction. I created alleles of tra1 which convert the terminal residue to serine (tra1-F3744S) or arginine (tra1-F3744R), an allele with the two terminal residues inverted (tra1-WF-FW), and alleles which resulted in deletions of one or two residues (tra1-Δ1and tra1-Δ2). tra1-WF-FW supported cell viability, whereas tra1-F3744S supported cell viability in the presence of the tti2-F328S suppressor. Slow growth at 37°C resulting from Tra1-WF-FW was also suppressed by tti2. Tra1-F3744S grew slowly at 37°C and on 6% ethanol in the presence of the suppressor. tra1-F3744R, tra1-Δ1, and tra1-Δ2 would not support viability in the presence or absence of the suppressor.
To better understand the structure and function of Tti2, truncation mutations were created to identify essential regions of Tti2. These truncation mutations were assessed by analyzing cell viability, protein expression levels, and interaction with Tti1 and Tel2, two other members of the TTT complex. Only Tti2-53-421 (containing residues 53-421) supported viability and retained its ability to interact with Tel2 and Tti1 at near wildtype levels. Tti2-53-238 and Tti2-1-238 interact with Tel2 but do not support viability. All of the other mutations did not support viability and showed minimal binding affinity to Tti1 and Tel2.
A terminal mutation of another PIKK family member, Mec1 (mec1-W2368A) also results in slow growth at 37°C. Interestingly, the tti2-F328S suppressor does not suppress the mec1-W2368A phenotype. Using bioinformatics approaches, I identified rpn3-L140P, encoding a component of the proteasomal cap, as a suppressor of mec1-W2368A.
Pillon, Samantha A., "Insights into the Function of the FATC Domain of Saccharomyces Cervisiae TRA1 via Mutation and Suppressor Analysis" (2013). Electronic Thesis and Dissertation Repository. 1549.