The long term goal of my research is to determine the mechanisms that control the initiation of protein synthesis in yeast The goal of this proposal is to describe in genetic and molecular terms components of the translation initiation complex that function in mediating ribosomal binding of mRNA, scanning of the leader region and recognition of the initiator region as well as the mechanisms by which these components may control the initiation process. The following specific aims are directed at achieving these goals: 1) Genetic reversion analysis has identified ten different suppressor genes which when mutated restore HIS4 expression despite a stem-loop translational barrier in the leader region. These as well as other suppressor genes, isolated for their ability to suppress stem-loop mutations at alternative positions in the leader, will be characterized to determine if their gene products function at the time of ribosomal binding or scanning of mRNA. 2) The functional significance of the 7methylGppp cap structure at the 5' end of mRNA for translation initiation in yeast will be investigated by genetic methods. The HIS4 gene has been put under the control of the rDNA promoter as an attempt to produce """"""""capless"""""""" HIS4 mRNA in yeast This rHIS4 mRNA will be characterized for the absence of a posttranscriptional modification at the 5' end of the message and rHIS4 strains will be used to identify genetic suppressors as potential candidates for gene products that might function in promoting ribosomal binding to mRNA. This genetic approach may also provide a productive way to explore alternative in vivo mechanisms for ribosomal binding to mRNA. 3) Characterization of the suil suppressor locus has shown that this gene product is functionally related to the sui2 and SUI3 suppressor locus but not a subunit of elF-2. As an attempt to gain insight into the function of suil we will use molecular and biochemical approaches to try to establish what gene products suil might interact with or if it is part of a complex with eIF-2 that is important for the start site selection process. In addition, we will continue with our characterization of recessive lethal, SUI suppressor mutants that we isolated in diploid cells to determine if other components of the initiation complex are essential for mediating start site selection. 4) The sui2 gene which encodes the alpha subunit of eIF-2 is phosphorylated at three other Ser residues by caesin kinase Il. We will now determine whether these phosphorylation events, or any other as yet uncharacterized phosphorylation events, are of any consequence to eIF-2 function in yeast or of any physiologic significance as a mechanism of controlling translation initiation. In light of similarities we have established among the yeast and human initiation processes and their translation factors, as well as a common translational control mechanism that is important for anti-viral response in humans, our studies of the basic translation initiation process in yeast should continue to prove valuable to fundamental control processes in all eukaryotes that are of biomedical importance.
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