THE FIDELITY OF PROTEIN-SYNTHESIS - CAN MISCHARGING BY ASPARTYL-TRANSFER RNAASP SYNTHETASE LEAD TO THE FORMATION OF ISOASPARTYL RESIDUES IN PROTEINS

We have tested the hypotesis 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 β-carboxyl group is linked to the tRNAAsp instead of the main chain α-carboxyl group. If this altered Asp-tRNAAsp is a substrate for the ribosomal elongation reactions, a polypeptide will be made with an isoaspartic acid, or β-linkage, in which the peptide chain is branched at the side chain of the aspartic acid residue. Using an ammonium sulfate fraction of aspartyl-tRNAAsp synthetase from Escherichia coli and [3H]aspartic acid, we have prepared [3H]aspartyl-tRNAAsp complexes and directly analyzed the linkage of the [3H]aspartate to the tRNA by identifying the products of ammonolysis. Normal attachment of the α-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 10000 isoasparagines. These results show that the bacterial aminoacyl-tRNA synthetase can very accurately distinguish between the α- and β-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. © 1990.