Asparaginyl-tRNA aminoacylation levels and asparagine synthetase expression in cultured Chinese hamster ovary cells.

Abstract

The regulation of asparagine synthetase in wild type Chinese hamster ovary cells and in several mutant strains containing defective aminoacyl-tRNA synthetases has been studied. When wild type cells are transferred from complete medium to medium lacking asparagine, the extent of aminoacylation of tRNA*“” decreases and asparagine synthetase activity increases. Similar results are observed when a mutant containing a temperature-sensitive asparaginyl-tRNA synthetase is transferred from permissive to restrictive conditions. Upon readdition of asparagine to wild type cells or transfer of mutant cells to permissive conditions, the extent of aminoacylation of tRNA*“” is rapidly increased and the activity of asparagine synthetase slowly decreases. An inverse correlation between the extent of tRNA*“” aminoacylation and the level of asparagine synthetase activity is observed when the mutant containing a defective asparaginyl-tRNA synthetase is grown in media containing a range of asparagine concentrations. Mutants with altered leucyl-, methionyl-, and lysyltRNA synthetases alr,o have elevated asparagine synthetase activity when grown under conditions that lead to decreased aminoacylation of their cognate tRNA species. Under these conditions tRNA*“” is fully aminoacylated. The kinetics of asparagine synthetase activity regulation is very similar under conditions of asparagine deprivation or restricted charging of tRNA’““. Experiments with cycloheximide and emetine suggest that the increased asparagine synthetase activity in the aminoacyl-tRNA synthetase mutants is not due to a decreased rate of protein synthesis in these cells. Therefore, elevated asparagine synthetase activity is correlated with decreased aminoacylation of several, if not all, tRNAs. This result seems to rule out a specific and direct mechanism based solely on the extent of aminoacylation of tRNA*“” in the regulation of asparagine synthetase levels in Chinese hamster ovary cells.