Characterization of the Interactions of Eukaryotic Initiation Factors, Ribosomes, and MRNA

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Goss 9600521 The long term goal of this project is to understand the mechanism of eukaryotic protein synthesis. Eukaryotes initiate translation differently in normal cell and in viral infecled cell or cells under stress conditions such as heat shock. The overall scheme of protein synthesis has been described, but the details and particularily the kinetics of this process remain to be elucidated. A detailed study of the equilibrium and kinetics of the interaction of eukaryotic initiation factors with mRNA and oligonucleotides is proposed. Direct fluorescence measurements will be used to elucidate the interaction of initiation factors and mRNA or oligonucleotides. The interaction of several initiation factors that bind to the 5' noncoding region have been previously analyzed. The kinetics of these interactions will now be determined. These multiple interactions will be analyzed to determine if the binding interactions are cooperative, anti-cooperative, or independent. Kinetic results will determine the rate limiting steps and the probable pathway of interactions. These results will aid in formulating a detailed molecular mechanism for the assembly of the eukaryotic initiation cornplex. %%% This project will elucidate the mechanism for protein synthesis in plant systems. Regulation of protein synthesis is important in regulation of cell growth and development as well as in biotechnology applications. Plants provide possible hosts for production of vacines and other proteins of commercial interest without the contaminants that may be present from a bacterial or other host used for gene cloning. lt is therefore important to understand the regulation of protein synthesis in plants. These studies will determine the rate limiting steps for interactions that determine the production of proteins. Fluorescence spectroscopy will be used to measure these interactions and determine the details of this process. By elucidating the rate limiting steps one can then determine the possible intervention points t o increase or decrease the rate of the overall protein synthesis process. ***