title: The fidelity of protein synthesis: can mischarging by aspartyl-tRNA(Asp) synthetase lead to the formation of isoaspartyl residues in proteins?
abstract: We have tested the hypothesis that isoaspartic acid residues in proteins can arise via errors that occur during protein synthesis. One such error involves a mischarging step in which the aspartic acid side-chain beta-carboxyl group is linked to the tRNA(Asp) instead of the main chain alpha-carboxyl group. If this altered Asp-tRNA(Asp) is a substrate for the ribosomal elongation reactions, a polypeptide will be made with an isoaspartic acid, or beta-linkage, in which the peptide chain is branched at the side chain of the aspartic acid residue. Using an ammonium sulfate fraction of aspartyl-tRNA(Asp) synthetase from Escherichia coli and [3H]aspartic acid, we have prepared [3H]aspartyl-tRNA(Asp) complexes and directly analyzed the linkage of the [3H]aspartate to the tRNA by identifying the products of ammonolysis. Normal attachment of the alpha-carboxyl group of aspartate to the tRNA produces [3H]isoasparagine, while the mischarging reaction leads to [3H]asparagine formation after ammonolysis. We have separated [3H]isoasparagine from [3H]asparagine and found an upper limit of 1 asparagine per 10,000 isoasparagines. These results show that the bacterial aminoacyl-tRNA synthetase can very accurately distinguish between the alpha- and beta-carboxyl groups of aspartic acid and suggest that only a very small fraction of the isoaspartic acid residues found to occur in cellular proteins may be the result of mischarging steps.
authors: Department of Chemistry and Biochemistry, University of California, Los Angeles 90024-1569.
authors: J A
authors: JA
authors: Momand
authors: S
authors: S
authors: Clarke
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001226:Aspartate-tRNA Ligase / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis*; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology*; D007536:Isomerism; D012346:RNA, Transfer, Amino Acyl / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D012346:RNA, Transfer, Amino Acyl; D001224:Aspartic Acid; D000604:Amino Acyl-tRNA Synthetases; D001226:Aspartate-tRNA Ligase