Translation Elongation Factor 1alpha (EF-1alpha) is a central protein involved in delivering aminoacyl-tRNAs to the ribosome and assuring incorporation of the appropriate amino acid during protein synthesis. While this protein is extremely abundant, the cell needs to modulate the activity or levels of EF-1alpha for normal cellular growth since inappropriate expression of EF-alpha results in transformation of cell lines and occurs in many carcinomas. Further, changes in the activity of EF-1alpha occur during aging and alter longevity. The goal of this project is to understand the mechanisms that regulate efficient and accurate translation. There are many different mechanisms to assure the accurate and efficient translation of an mRNA, and it is clear these mechanisms converge on the action of EF-1alpha. The central role of EF-1alpha in elongation and its interactions with proteins and RNAs that modulate its activity are a very important factor in this process. Our previous studies support the hypothesis that changes in translational fidelity in cells harboring mutations in EF-1alpha are a result of many different effects on gene expression in vivo. Current models of elongation predict these include indirect effects that result from changes in the pool of active EF-1alpha x GTP and thus the occupancy of the A-site of the ribosome. However, EF-1alpha also directly affects this process through the presentation of aa-tRNA to the ribosome, the determination of cognate codon-anticodon interactions, and the hydrolysis of GTP required for kinetic proofreading. The use of the yeast Saccharomyces cerevisiae allows analysis of the translation elongation cycle both in vivo and in vitro. This proposal describes a set of biochemical studies of mutations predicted or designed to analyze both direct and indirect effects of EF-1alpha on accurate gene expression. Purification and analysis of wild-type and mutant EF-1alpha proteins in the partial reactions of translation elongation will determine the causes of the in vivo translation and growth phenotypes of these mutations. Furthermore, this information is utilized to apply genetic approaches to identify and characterize factors that modulate EF-1alpha activity; the guanine nucleotide exchange factor (EF-1Beta) and the GTPase activating factor (the ribosome). This analysis will also identify novel factors that affect or regulate acurate elongation. This coordinated approach is a unique method to extend our understanding of the many EF-1alpha dependent steps required for accurate and efficient gene expression.
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