The long term goal of this 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, molecular, and biochemical terms components of the translation initiation complex that function in mediating and controlling ribosomal recognition of the initiator region. The PI has implicated the sui1 suppressor gene, indirectly, to participate in the GTP hydrolysis step during the AUG start site selection process. Now he plans to determine the function of Sui1, as a subunit of eIF-3, in the GTP hydrolysis/initiator-tRNA dissociation steps by combining the genetics with biochemical studies. He has recently shown that the beta and gamma subunits of eIF-2 as well as eIF-5 function in controlling ribosomal recognition of an AUG codon through a GTP hydrolysis switch. He will now determine the function and interactions of the alpha-subunit of eIF-2 in this process using multi- pronged approaches. He has recently identified a number of extragenic suppressors that improve the growth properties of a sui3 ts mutant at the restrictive temperature. One of these genes encodes the yeast homologue of the bacterial translation initiation factor IF2. He will determine the in vivo and in vitro function of yeast IF2 in translation initiation as well as other genetic suppressors that may be related to IF2 function in yeast. All of the SUI suppressor mutants he has identified preferentially initiate translation at UUG codon when no AUG codon is present at HIS4. Suppressor mutants that can initiate preferentially at a GUG codon will be selected. These suppressor mutants might enable him to identify those gene products that sense the proper three base codon:anticodon interaction during translation initiation or which normally negatively control the initiator-tRNA (3'-UAC-5') to form a G:U base pair in the +1 position. The PI hopes to apply the translation initiation process in vitro with defined proteins. In light of similarities established among the yeast and human initiation components and processes as well as common translational control mechanisms, these studies may prove useful in the study of fundamental control processes in all eukaryotes that are of biomedical importance.
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