Subunit Function and Topology in Glycyl-Trna Synthetase

Nagel, Glenn

Abstract

This research explores the function and organization of the two types of subunits which comprise the enzyme glycyl-tRNA synthetase (Alpha2 Beta2). The existence of dissimilar subunits is rare in this class of enzymes and the present work is aimed at understanding how and why they are used in the biological role of the enzyme, the aminoacylation of transfer RNA, and in a proposed new role, the regulation of its structural gene, glyS. Specifically, electron microscopic and electrophoretic studies on cross-linked samples of the enzyme will be used to refine our model for the enzyme's overall structure and subunit topology. These data will provide a firm basis for understanding and synthesizing data on the function of its subunits. Site-specific modification reagents will be used to chemically label binding sites on the enzyme for ATP (glycine activation site) and for the 3' terminus of tRNA (glycine esterification site) as well as a region interfacing with the acceptor stem of the tRNA molecule. These studies will allow the localization and quantitation of sites on one or the other type of subunit. Additionally, the isolation of labeled peptides from the modified enzymes will allow us to begin mapping functional domains incuding nucleic acid-recognition regions in the known amino acid sequence. Subunit-specific antibodies will also be employed to probe the location of these sites and to ask experimentally whether related enzymes are structurally homologous to glycyl-tRNA synthetase. Finally, direct binding and nuclease protection experiments using small (less than 900 bp) DNA fragments containing the glyS promoter region and purified glycyl-tRNA synthetase will explore the strength and specificity of the interaction and the exciting possibility of autoregulation of glyS by its gene product. Parallel studies on alanyl-tRNA synthetase and alaS DNA, a known autoregulatory system, will be completed for comparison and to better characterize the alaS regulation. Taken together, these studies constitute the first systematic study of subunit function in a rare Alpha2 Beta2) aminoacyl-TRNA synthetase. These studies should augment greatly our understanding of the mechanism and regulation of a fundamental and essential reaction of protein biosynthesis.