Abstract
Glycyl-tRNA synthetase is one of two aminoacyl-tRNA synthetases in Escherichia coli that is comprised of heterologous subunits which are organized in an alpha 2 beta 2 quaternary structure. The two subunits are encoded by a single mRNA with the region for alpha (303 codons) subunit followed by that for beta (689 codons) subunit. Five COOH-terminal deletions in the beta subunit coding region have been created. Each deletion protein has been investigated for its synthesis and stability in vivo, adenylate synthesis activity in vitro, and aminoacylation activity in vivo and in vitro. This has been done in the presence of free alpha subunit and, additionally, with alpha subunit that is fused by its carboxyl terminus to the amino terminus of each of the beta subunit deletion proteins. With a fused or unfused alpha chain, over 100 amino acids can be deleted from the carboxyl terminus of the beta chain without loss of in vivo complementation of a delta glyS (deletion) strain. Further analysis shows that the alpha subunit and approximately the amino-terminal half of the beta subunit are sufficient for the adenylate synthesis activity. In particular, a deletion of 306 amino acids from the COOH terminus of the beta subunit has little effect on the Km parameter for ATP or glycine in the pyrophosphate exchange reaction. The tRNA-dependent step in aminoacylation requires additional beta subunit sequences on the COOH-terminal side of those needed for adenylate synthesis. In these respects, the functional organization of the beta chain parallels that of several aminoacyl-tRNA synthetases which have only homologous subunits. In the case of the glycine enzyme, however, the heterologous alpha subunit is required for the elucidation of activities encoded by functional determinants of the beta chain.
Highlights
Glycyl-tRNA synthetase is one of two aminoacyltRNA synthetases in Escherichia coli that is comprised of heterologous subunits which are organized in an a&. quaternary structure
Each deletion protein has been investigated for its synthesis and stability in vivo, adenylate synthesis activity in vitro, and aminoacylation activity in vivo and in vitro
This has been done in the presence of free a subunit and, with
Summary
The a6575 and a6383 deletions result from the elimination bf 114 and 306 codons, respectively, from the COOH-terminal p subunit coding sequences by the removal of appropriate restriction fragments. All of these deletions encode extra COOH-terminal amino acids that result from translation of vector sequences. Two COOH-terminal deletions (ap511 and o/3475) had stop codons (ochre) inserted at the designated positions by oligonucleotide-directed mutagenesis of an mp vector containing the glyS gene [13]. TAA stop codon and the p subunit’s ATG initiator codon changed to GAA (Gin) and GCA (Ala), respectively This results in a Fusion 3 glvcvl-tRNA svnthetase that has the COOH terminus of the Other Assays-Protein concentration was determined by the dye binding method (Bio-Rad)
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