Date of Award

January 1974

Degree Type

Dissertation/Thesis

Abstract

This study was undertaken to determine whether the formation of methionyl-, prolyl-, and threonyl-tRNA synthetases is "constitutive" in nature, or subjected to control by an amino acid-specific mediated repression-like mechanism. Using auxotroph for methionine, proline, and threonine and employing shifts from excess to limiting concentrations of the respective amino acid, the net change of the cognate aminoacyl-tRNA synthetase activity, as measured by its specific activity, was determined. Proline and threonine-restricted growth caused a 3 to 4-fold derepression of the differential rate of synthesis of the prolyl- and threonyl-transfer ribonucleic acid (tRNA) synthetases, respectively. Similarly, there was approximately a 20-fold derepression in the rate of synthesis of methionyl-tRNA synthetase during methionine restriction. Addition of the respective amino acids to such derepressed cultures resulted in a repression of synthesis of the cognate synthetase. By the use of increasing amounts of the respective amino acids, evidence was provided which indicated that the rate of synthesis of the cognate aminoacyl-tRNA synthetase was inversely proportional to the amino acid concentration used. The control of methionyl-tRNA synthetase was further studied in methionyl-tRNA synthetase mutants of Escherichia coli and Salmonella typhimurium. The results of activity determinations using crude extracts indicate that this enzyme of E. coli mutant strain possessed a reduced affinity for methionine tRNA; while this enzyme of a S. typhimurium mutant exhibited a decreased affinity for L-methionine. Results of P22 transduction suggest that the mutation in both strains was in metG, the structural gene for methionyl-tRNA synthetase. The differential rate of methionyl-tRNA synthetase formation in these two mutants was several-fold greater than that of the respective parental strains. On the other hand, the level of in vivo aminoacylation of methionine tRNA was only about one-third that of the parent strains. In addition, the steady state synthesis of this enzyme in a metG + strain was enhanced by inhibition of formylation of charged methionyl-tRNA. These results support previous findings, and suggest that synthesis of methionyl-, prolyl-, and threonyl-tRNA synthetases is regulated by a repression-like mechanism. These results further suggest that aminoacylation of methionine tRNA is a necessary step in repression control of methionyl-tRNA synthetase of both E. coli and S. typhimurium strains.

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