Mitochondria, whether in lower or higher eukaryotes, are endowed with the capability to translate a small number of proteins encoded by their genomes. With the exception of the transfer and ribosomal RNAs, and a single ribosomal protein, whose genes are located in mitochondrial DNA, all other components of the translational machinery are derived from nuclear genes. During the past grant period, nuclear respiratory deficient mutants of yeast affected in various aspects of mitochondrial metabolism have been isolated and characterized. Among this collection, approximately 55 complementation groups have been ascertained to be impaired in mitochondrial protein synthesis. This subset of mutants has allowed characterization of different mitochondrial aminoacyl-tRNA synthetases, ribosomal proteins, and translational initiation and elongation factions. The analysis of the aminoacyl-tRNA synthetase genes have been particularly fruitful in revealing new and unsuspected evolutionary relationships between different members of this enzyme family.The studies embodied in the present proposal will provide addition insights into the nature of the mitochondrial translation system through continued biochemical and genetic analyses of respiratory deficient yeast mutants. Several objectives will be pursued in the course of these investigations. First, the PI intends to complete the characterization of other mitochondrial aminoacyl-tRNA synthetases and to start structural studies on the lysyl-tRNA synthetase. This information will be used to further assess the evolutionary derivation of this multigene family. A second important goal is to delineate the factors responsible for regulating the expression of mitochondrial synthetases and to clarify the biochemical rationale for the existence of separate genes for some of the mitochondrial and cytoplasmic homologues. Finally they expect to work out an in vitro system of mitochondrial protein synthesis which will permit more incisive studies on the mechanism by which the products of nuclear genes such as CBP6, CBP7 (CBS2), and CBS1 facilitate translation of the cytochrome b mRNA and pre-mRNA.
