The objectives of this proposal are to identify cellular components involved in ribosomal selection of the translation initiation codon in the yeast Saccharomyces cerevisiae. A combination of the powerful techniques of classical yeast genetics and modern molecular biology will be used to characterize revertants of a previously defined mutant (cycl-362 allele) known to be defective in initiation of translation of CYC1 messenger RNA (Stiles et al., Cell 25:277 (1981)).
Specific aims i nclude the following: (i) isolate revertants of cycl-362 by established selection procedures; (ii) genetically distinguish local (CYC1-linked) from extragenic suppressors; (iii) characterize the CYC1 locus from each by DNA sequence analysis and 5' endpoint transcript mapping; (iv) clone the most appropriate extragenic suppressor of the cycl-362 defect; (v) extensively characterize the cloned suppressor; and (vi) ultimately identify the suppressor-encoded trans-acting factor. Extensive biochemical studies of the translational apparatus has identified the components involved in translation initiation and elongation. However, these results do not account for potential control of gene expression at the translational level. It is clear, however, that a number of genes, including many involved in physiological and developmental control mechanisms, are translationally regulated. In yeast, for example, a nuclear gene involved in coordinate control of amino acid biosynthetic genes is translationally controlled. In higher eukaryotes, genes involved in the heat shock response, light-induced developmental signals, and other processes are translationally regulated. Moreover, recent evidence suggests that the tat gene product from human immunodeficiency virus controls translation of the viral encoded messenger RNAs. Translational control is clearly a significant mechanism for controlling the expression of certain genes, yet our present understanding of translation initiation is insufficient to account for such mechanisms. The research program described herein is designed to address this problem by specifically characterizing the factors involved in selection of the translation initiation codon.
