ABSTRACT
We have isolated temperature resistant revertants from temperature sensitive E. coli strains containing either a thermolabile glutaminyl-tRNA synthetase or leucyl-tRNA synthetase. Among the revertants which still contained the thermolabile leucyl-tRNA synthetase we found two classes of regulatory mutants (leuX and leu Y) which have elevated levels of this enzyme. The leuX mutation specifies an operator-promoter region adjacent to the structural gene (leuS) for the enzyme. The leuY gene maps away from the leuS gene and codes for a protein. Using these mutants we demonstrated that the levels of leucyl-tRNA are related to the derepression of the leucine and isoleucine-valine operons. Among the revertants which still contained the thermolabile glutaminyl-tRNA synthetase were characterized three classes of mutants, glnT, glnU, and glnR. The glnT and glnU mutants contain elevated levels of tRNAgln, while the glnR mutant possesses elevated levels of glutaminyl-tRNA synthetase. The level of glutamine synthetase, the enzyme responsible for the formation of glutamine, is also derepressed in the glnT and glnR mutants.
Subject(s)
Amino Acyl-tRNA Synthetases/biosynthesis , Escherichia coli/genetics , Gene Expression Regulation , RNA, Transfer/biosynthesis , Amino Acyl-tRNA Synthetases/genetics , Chromosome Mapping , Genes, Regulator , Glutamate-Ammonia Ligase/genetics , Leucine-tRNA Ligase/genetics , Mutation , Operon , RNA, Transfer/genetics , RNA, Transfer, Amino Acyl/genetics , TemperatureABSTRACT
The influence of buffer, pH, Mg2+, ATP, Na+, K+ and temperature on the extent and rate of aminoacyl-tRNA synthesis was studied with the shark Mustelus mento liver tRNAs and aminoacyl-tRNA synthetases. The optimum pH for the aminoacylation of tRNAMet was 8.3 and for tRNAVal 7.0. For these two tRNAs the Mg2+ optimum was related to the levels of ATP required and to the pH of the reactions. It is suggested that the Mg2+ concentration required for each aminoacylation system reflects the true conditions under which the substrate for the enzyme, the MgATP2- complex, is formed. The effect of monovalent cations was also examined. Val-tRNA synthesis was slightly stimulated up to a concentration of 50 mM NaCl (KCl). Over 100 mM salt, a rapid inhibition was observed. Met-tRNA synthesis behaves differently by being stimulated over a wide range of salt concentrations.