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

Andrulis, I.L.; Hatfield, G.W.; Arfin, S.M.

Journal of Biological Chemistry 254(21): 10629-10633

1979


ISSN/ISBN: 0021-9258
PMID: 40971
Accession: 068518740

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Abstract
The regulation of asparagine synthetase in wild type Chinese hamster ovary (CHO) cells and in several mutant strains containing defective aminoacyl-tRNA synthetases was studied. When wild type cells are transferred from complete medium to medium lacking Asn, the extent of aminoacylation of tRNAAsn 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 Asn to wild type cells or transfer of mutant cells to permissive conditions, the extent of aminoacylation of tRNAAsn is rapidly increased and the activity of asparagine synthetase slowly decreases. An inverse correlation between the extent of tRNAAsn 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 Asn concentrations. Mutants with altered leucyl-, methionyl-, and lysyl-tRNA synthetases also have elevated asparagine synthetase activity when grown under conditions that lead to decreased aminoacylation of their cognate tRNA species. Under these conditions tRNAAsn is fully aminoacylated. The kinetics of asparagine synthetase activity regulation is very similar under conditions of asparagine deprivation or restricted charging of tRNALeu. 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. Elevated asparagine synthetase activity is correlated with decreased aminoacylation of several, if not all, tRNA. A specific and direct mechanism based solely on the extent of aminoacylation of tRNAAsn in the regulation of asparagine synthetase levels in CHO cells is ruled out.