Master of Science
Dr. Jim Karagiannis
The phosphorylation status of the largest sub-unit of RNA polymerase II (Rpb1p) is crucial to the control of transcription in eukaryotes. The domain subject to this phosphorylation is known as the carboxyl terminal domain (CTD) and consists of multiple repeats (from 20 to 52 copies depending on the species in question) of the heptad sequence Y1S2P3T4S5P6S7. Interestingly, differential phosphorylation of S2, S5, and S7 residues is known to play an important role in the control of pre-mRNA processing. To determine the number of CTD repeats required for viability in Schizosaccharomyces pombe, truncated CTD constructs were integrated into the S. pombe genome through homologous recombination. I have observed that a minimum of eight heptad repeats is required for normal growth. While five heptad repeats is sufficient for viability, strains bearing these constructs exhibit impaired growth as well as morphological abnormalities. Next, in order to study the phenotypic effects of altered phosphorylation patterns, site-directed mutants were created in which alanine residues replaced serine (mimicking the non-phosphorylated state) or in which glutamate residues replaced serine (mimicking the phosphorylated state). Interestingly, alteration of the phosphorylation status of both S5 and S7 residues resulted in a variety of pleiotropic defects related to both cytokinesis and morphogenesis. Such a role for S5 and S7 phosphorylation is further supported by genetic analysis demonstrating synthetic genetic interactions between CTD site mutants and mutants affecting the function of the cell division machinery.
Hoffman, Kyle S., "Phenotypic Analysis of Schizosaccharomyces Pombe Strains bearing Site-Directed Mutations in the Carboxy Terminal Domain of the Largest Subunit of RNA Polymerase II" (2012). University of Western Ontario - Electronic Thesis and Dissertation Repository. Paper 765.