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

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9722907 Goss The long term goal of this project is to understand the mechanism of eukaryotic protein synthesis. Eukaryotes initiate translation differently in normal cells than in virus infected cells or cells under stress conditions such as heat shock. The overall scheme of protein synthesis has been described, but the details and particularly the kinetics of this process remain to be elucidated. The equilibrium and kinetics of the interaction of eukaryotic initiation factors (eIFs) with mRNA and oligonucleotides will be examined. The effects of eIFs on ribosome binding to several oligonucleotides and mRNA will be determined. Direct fluorescence measurements will be used to elucidate the interaction of initiation factors, ribosomes and mRNA or oligonucleotides. 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. Quantitative data will give insight into the effect of mRNA secondary structure on ribosome binding, the possible communication between the 5' and 3' termini of RNA, and the possibility of internal ribosome entry in higher plant systems. These results will aid in formulating a detailed molecular mechanism for the assembly of the eukaryotic initiation complex. 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 vaccines and other proteins of commercial interest without the contaminants that may be present from a bacterial or other host used for gene cloning. It 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. By elucidating these steps o ne can then determine the possible intervention points to increase or decrease the rate of the overall protein synthesis process.