The long-term objective of this proposal is to investigate the mechanism of protein synthesis in eucaryotes. Protein synthesis is a fundamental process in all organisms and must be understood to fully characterize normal cell physiology. Such knowledge is clearly important in understanding the mechanisms of disease. In addition, since translation is often a target for drugs, knowledge of protein synthesis should help elucidate the mechanism of action of certain drugs. The yeast Saccharomyces cerevisiae is a promising organism to use to investigate the eucaryotic translational apparatus. Studies of yeast ribosomes will probably be relevant to an understanding of the ribosomes of higher eucaryotes due to considerable similarities. The unique advantage of yeast is the availability of the techniques of classical genetics, gene cloning and transformation. The overall plan is to obtain and characterize mutations in the translational apparatus of Saccharomyces cerevisiae. The isolation of revertants of these mutations will allow the identification of still more involved loci, and will give insight into the interaction of the various parts of the protein synthesizing machinery. As a first step in determining what cellular components correspond to these mutations, the genes will be cloned. The products of the cloned mutant genes can then be isolated and studied. Eventually, these products could be characterized by using an in vitro protein synthesis system with yeast components. Specifically, mutants which affect translational accuracy in yeast will be isolated by selecting for new omnipotent-suppressors (suppressors which cause general translational misreading), selecting for mutations which enhance (allosuppressors) or reduce (antisuppressors) the efficiency of the omnipotent-suppressors, selecting for revertants of antisuppressors, and selecting for drug dependent mutants. Genetic analyses of these mutants would be followed by the cloning of selected single genes affecting transalational accuracy. The clones will be used to map the genes and isolate homologous mRNA and the encoded protein. Structure and function relationships of the protein would be examined by DNA sequencing of alleles with different phenotypes.
