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.
Highlights
Chinese hamster ovary cells and in several mutant strains containing defective aminoacyl-tRNA synthetases has been studied
We have measured the activity of asparagine synthetase in relation to the fraction of aminoacylated tRNA*“” and we have studied the regulation of asparagine synthetase activity in several other mutants with defects in specific aminoacyl-tRNA synthetases [23,24,25]
The initial suggestions of roles for tRNA in the regulation of various aspects of cellular physiology in animal cells were based upon the examination of variations in cellular content of isoacceptor tRNAs
Summary
Chinese hamster ovary cells and in several mutant strains containing defective aminoacyl-tRNA synthetases has been studied. In 1965, Eidlic and Neidhardt [1] reported a correlation between decreased aminoacylation of tRNA and increased expression of amino acid biosynthetic enzymes This suggestion was based on the observation that growth of a mutant. Studies with amino acid analogs and temperaturesensitive aminoacyl-tRNA synthetase mutants of animal cells have suggested that tRNAs are involved in a number of other cellular functions. If the role of tRNA in the regulation of amino acid biosynthesis in animal cells is comparable to that in bacterial cells, an inverse correlation between the extent of aminoacylation of tRNA*“” and asparagine synthetase activity in uiuo would be expected [21, 22]. Elevated levels of asparagine synthetase activity are observed when the aminoacylation of several other tRNAs is restricted
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