REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS INITITATION

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Protein synthesis plays a central role in growth and differentiation, response to change, and overall energy utilization of the cell. Viruses utilize the host translational machinery, and host cells have evolved complex antiviral responses. Deregulation of protein synthesis leads to malignant transformation. This project seeks to understand the rate- limiting step of protein synthesis, recruitment of mRNA to the ribosome, where much of translational control occurs. Two key factors in this process are eIF4E, the mRNA cap-binding protein, and eIF4G, a protein which links together the 40S ribosome, eIF4E, and the RNA helicase eIF4A. The roles of the eIF4 initiation factors will be studied by asking the following questions: 1. How do the eIF4 factors participate in recruitment of mRNA to the ribosome? We will arrest of intermediates of initiation complex assembly using allyloligonucleotides, overexpress eIF4G and a protease- resistant variant in cultured cells to determine oncogenic phenotype, test the effect of specific functional domains of eIF4G, and characterize eIF4E from C. elegans with respect to binding to m7G- and m3(2,2,7) G-containing caps. How does the phosphorylation of eIF4E affect protein synthesis? We will express in CREF cells eIF4E variants around the phosphorylation site to determine phenotype, examine the characteristics of eIF4E phosphorylation by PKCzeta, determine the properties of phosphorylated eIF4E by biophysical methods, and test whether eIF4E participates in a phosphorylation cycle during initiation. What physiological factors determine the intracellular levels and activity of eIF4G? We will study eIF4G synthesis, degradation and steady-state levels as a function several parameters as well as temperature-dependent conformational changes in recombinant eIF4G by biophysical and biochemical. How does the IRES of eIF4G mRNA direct internal initiation? We will delineate the borders of the eIF4G IRES, determine its secondary structure, characterize its translation in vivo, and attempt to find conditions for its utilization in vitro. And finally, what is the structure of the eIF4E gene? We will complete determination of the gene structure, define the promoter, and attempt to identify cis-acting regulatory elements.