It is proposed that a study of the regulation, synthesis, and expression of eukaryotic tRNA can be accomplished in yeast - a simple eukaryote in which combined genetic and biochemical investigations can be conducted. The research is directed towards isolating mutants or establishing conditions for which tRNA synthesis or expression is aberrant. Two approaches to accomplishing this are described: (1) isolation of mutants which conditionally lose the ability to suppress nonsense mutations and also fail to grow at the nonpermissive temperature (the types of mutations which cause cells to lose suppressor activity will be studied by reconstructing suppressor stains bearing mutations in genes affecting well described aspects of yeast cells growth); (2) tritium suicide of cells with functional RNA polymerase III. The mutants will be analyzed genetically to determine the number of complementation groups and to determine whether the mutations affect more than one species of tRNA. The RNA from the cells will be analyzed biochemically to establish a pathway of processing events, to determine the role of modified bases in tRNA function, and to study the subunits of RNA polymerase III.
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