A thorough understanding of the mechanism of protein synthesis is valuable because of its importance in cellular metabolism and because gene expression is controlled in part at the level of translation. The broad goals of the project are to elucidate the mechanisms of initiation and control of protein synthesis in mammalian cells. We shall study the structure and function of the initiation factors by two basic approaches: analyses in vitro with highly purified factors; and characterizations of crude cell lysates which mimic in vivo states. Using purified factors, we shall elucidate how mRNAs are recognized by the translational machinery, determine the structure of eIF3 by protein chemical techniques, study factor-ribosome interactions by fluorescence and cross-linking techniques, and construct active in vitro systems for protein synthesis dependent on the factors. We shall use specific antibodies against the factors to inhibit protein synthesis in lysates to show that each factor is required for protein synthesis. Initiation factors in crude cell lysates will be characterized by immunoblotting and 2-dimensional polyacrylamide gel electrophoretic techniques to measure their levels, molecular weights and extent of covalent modification, e.g., by phosphorylation. We shall correlate changes in the molecular parameters with modulations in in vivo rates of protein synthesis by examining cells in different physiological states, such as in serum or amino acid deprivation or during mitosis, and seek evidence for control through factor modification. Strong correlations will be studied in greater detail in vitro with purified, modified factors. How factor levels are regulated will be studied by cloning cDNA sequences homologous to factor genes and studying factor gene expression. We also shall examine how poliovirus causes the degradation of the cap binding protein complex, how the cytoskeleton may be involved in protein synthesis, and how mRNPs are mobilized into active polysomes. The interplay of in vitro studies involving crude and purified preparations will help avoid artifacts inherent in such studies and should provide detailed molecular mechanisms of translational control.
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