CONTROL OF EIF-2 ACTIVITY IN REGULATION OF TRANSLATION

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The quantitative control of translation is an important mechanism in the short-term regulation of gene expression. It is achieved by changes in the rate of mRNA binding to ribosomes and seems to be regulated at the level of initiator RNA interaction with the small ribosomal subunit. Eukaryotic initiation factor 2, eIF-2, promotes this step of the initiation sequence and is found to be reversibly phosphorylated in circumstances in which the initiation of protein synthesis is reduced. The mechnism which phosphorylation of eIF-2 reduces the rate of initiation is not completely understood, but involves the perturbation of the recycling mechanism which promotes guanine nucleotide exchange on the factor. Although the importance of eIF-2 phosphorylation has been clearly demonstrated in the regulation of protein synthesis in reticulocytes and reticulocyte lysate, it is not known to what extent these regulatory mechanisms represent general mechanisms that may be found in a variety of cell types and under varying physiological conditions. The present proposal undertakes to complete the investigations on the mechanisms by which phosphorylation reduces eIF-2 activity and to work out the overall activity cycle of eIF-2 in reticulocyte lysate. These studies will utilize antibodies to eIF-2 and its recycling protein, eIF-2B. The other major question to be addressed is how important a role eIF-2 plays in the regulation of protein synthesis in animal cells. The antibodies will expedite the investigation of eIF-2 metabolism in cells other than reticulocytes and facilitate the assessment of the extent to which modification of eIF-2 is employed in other cell types. A series of cultured rabbit cell lines will be used to document eIF-2:ribosome and eIF-2:eIF-2B ratios with respect to cell type and proliferation rate under steady state conditions. In addition, the association of eIF-2 with other components of the translational apparatus will be determined under changing nutrient environments. Finally isolated rat hepatocytes will be used to assess eIF-2 involvement in the regulation of protein synthesis by changing metabolite levels and hormonal signals.