Enzymology and Control of Amino Acid Activation

University Of Miami School Of Medicine, Coral Gables FL

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Abstract

DESCRIPTION (provided by applicant): The ultimate expression of the genetic makeup of a cell resides in its proteins. Protein biosynthesis is a complex process that requires the participation of numerous proteins and nucleic acids. A major determinant of the specificity of the process resides in its initial step, the activation and transfer of an amino acid to its cognate transfer RNA by an aminoacyl-tRNA synthetase. Since aminoacyl-tRNA formation ultimately depends on the availability of functional tRNAs and of functional aminoacyl-tRNA synthetases, it is the long range goal of this work to elucidate the pathways, enzymes and regulatory processes that determine the levels and activities of each of these components in cells. This includes analysis of the structure, function and mechanism of the ribonucleases originally identified for their involvement in tRNA biosynthesis, and elucidation of how the mammalian translation system is organized and how it actually uses aminoacyl-tRNA in vivo. The studies we propose should provide essential information in these central areas of macromolecular metabolism. The specific aims of this project are: 1) elucidation of the remaining unidentified rRNA maturation pathways; 2) determination of the physiological role of E. coli exoribonucleases; 3) elucidation of exoribonuclease active sites, substrate recognition and mechanism of action; 4) examination of RNases and RNA degradation during stress; 5) analysis of the organization of translation and the role of the actin cytoskeleton; 6) determination of the synthesis and function of the two forms of mammalian arginyl-tRNA synthetase; and 7) examination of nuclear protein synthesis and assembly of the mammalian translation machinery. In view of the central role of proteins in all aspects of cell function, a detailed understanding of how the components that participate in protein biosynthesis are made and how they actually function in the cell is of prime importance. Elucidation of these aspects of protein synthesis in normal cells is a prerequisite for a complete understanding of abnormal gene expression which may occur under a variety of pathological conditions.