Little is known about tRNA transcription and maturation in eukaryotes. Yeast is a simple eukaryote for which both genetic and biochemical studies can be conducted and for which suppressor tRNAs have been demonstrated. Therefore, combined genetic and biochemical studies, similar to those which have been successful in delineating the step involved in functional tRNA formation in prokaryotes, should be possible to carry out in yeast. This research is directed towards obtaining mutants of yeast which are conditionally defective in the accumulation of tRNA and in nonsense suppression. Three approaches to obtaining such mutants will be attempted: (1) Screening of already existing yeast mutants which are temperature sensitive for growth; (2) Tritium suicide of cells with functional RNA polymerase III; and (3) Selection of antisuppressor mutations. The mutants will be analyzed to determine the number of genes which are involved in tRNA production and to establish a crude pathway of maturation. The RNAs from the putative mutants will be analyzed by polyacrylamide gel electrophoresis to determine whether the mutant phenotypes result from transcriptional or post transaction studies. Putative mutants which are blocked in tRNA maturation will be studied to determine the size of the tRNA precursors and whether the RNA transcripts are monomeric or polymeric. If mutants defective in RNA polymerase or in the maturation of an unique RNA are obtained, they will be useful in future experiments, for establishing an assay for the fidelity of in vitro RNA transcription.