Characterization of temperature-sensitivity and its intragenic suppression in Saccharomyces cerevisiae tRNA nucleotidyltransferase mutants

• Main Author: Goring, Mark
• Format: Others
• Published: 2011
• Online Access: http://spectrum.library.concordia.ca/7532/7/Goring_MSc_S2011.pdf; Goring, Mark <http://spectrum.library.concordia.ca/view/creators/Goring=3AMark=3A=3A.html> (2011) Characterization of temperature-sensitivity and its intragenic suppression in Saccharomyces cerevisiae tRNA nucleotidyltransferase mutants. Masters thesis, Concordia University.

ATP:CTP-specific tRNA nucleotidyltransferase enzymes catalyze the stepwise addition of one CMP, a second CMP, and finally an AMP as needed to generate the functional 3’ termini of tRNA molecules. The following work concerns the structural and functional characterization of a temperature-sensitive variant E189F of tRNA nucleotidyltransferase from Saccharomyces cerevisiae, and two suppressor variants; R64WE189F and R64WE189K of this phenotype. Previous work has shown that E189K, E189F, and other variants of tRNA nucleotidyltransferase with substitution mutations at E189 are temperature-sensitive, and can exhibit enzymatic activity levels as low as 4% relative to that of native enzyme (Aebi et al., 1990, Shan et al., 2008). Moreover, E189F and E189K display almost identical levels of compromised thermal stability as compared to the native enzyme. Here, tRNA nucleotidyltransferase from recently isolated intragenic suppressor mutants of the temperature-sensitive phenotype are characterized in an effort to understand the development of the temperature-sensitive phenotype and its suppression. The suppressor strains R64WE189F and R64WE189K show a level of thermal stability that is comparable to E189F, or intermediate to E189F and E189 respectively. However, both suppressors display native-like levels of activity. It is evident that severely reduced activity is the major determining factor of the temperature-sensitive phenotype, not compromised thermal stability, and that a marked increase in specifc activity is responsible for its suppression. Consequently, these data also suggest that the temperature-sensitive phenotype may be caused by an inability of temperature-sensitive variants to meet the demand for mature tRNA at the restrictive temperature.