The fidelity of protein synthesis: can mischarging by aspartyl-tRNA(Asp) synthetase lead to the formation of isoaspartyl residues in proteins?
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.
Momand
J A
JA
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024-1569.
Clarke
S
S
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
D001426:Bacterial Proteins; D012346:RNA, Transfer, Amino Acyl; D001224:Aspartic Acid; D000604:Amino Acyl-tRNA Synthetases; D001226:Aspartate-tRNA Ligase