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Abstract
This dissertation explores the role of three enzymatic reactions involved in the transfer of the simple methyl group from S-adenosyl methionine in biological systems; two of these studies relate to different protein methylation reactions. One examines methylation on a nitrogen atom of a histidine residue in actin and the other pertains to a reversible methylation reaction on the oxygen atom of the carboxylic group at the C-terminus of protein phosphatase 2A (PP2A). The third methylation reaction studied here catalyzes methylesterification of nucleobases on tRNA nucleobases.
In my first study, the methylation state of His-73 on actin protein was examined in both the yeasts Saccharomyces cerevisiae and Candida albicans. I discovered that the conserved methylation of His-73, which had been found in almost all organisms, is not present in the actin of these yeast strains. The analysis of how yeast actin functions in the absence of this modification has allowed for a better understanding of the role of methylation on other actin species. In the second project, I identified a methyltransferase, (Ppm1) in yeast cells that modifies protein phosphatase 2A (PP2A) that in vivo participates in reversible modification of this important signaling enzyme.
The bulk of this dissertation focuses, however, on the third biological methylation reaction, the methylesterifcation of tRNA. Using S. cerevisiae as a model organism, I identified an enzyme, designated Trm9 (transfer RNA methylase), responsible for methylesterifying two nucleobases on tRNA that make up the majority of methyl esters in the yeast S. cerevisiae . In addition, I examined the physiological roles that Trm9 could play in cellular processes such as protein degradation and translation. Furthermore, the stability of the methyl ester species of tRNA was studied both in vivo and in vitro. Finally, in this thesis, the possible presence of a mammalian homolog of TRM9 is explored.
