Fidelity of Aminoacylation - Protein Synthesis

Fidelity of Aminoacylation

The aminoacyl-tRNA synthetases are remarkable enzymes since they recognize amino acids and their cognate tRNA molecules and join them together. Inaccuracies in either recognition process could be highly deleterious because choosing the wrong amino acid or the wrong tRNA would ultimately yield a protein with an incorrect sequence. We know,

however, from measurements on peptides highly purified from proteins of known sequence, that the overall frequency of misincorporation, at least of charged amino acids, is only about 1/1000.

Let us first consider the process of choosing the correct amino acid. The greatest difficulty in accurate translation appears to be in discrimi-nating between two highly similar amino acids. Valine and isoleucine are an example since replacing a hydrogen on valine with a methyl group yields isoleucine (Fig. 7.3). The valyl-tRNA synthetase should not have trouble in discriminating against isoleucine because isoleucine is larger than valine and probably does not fit into the active site on the enzyme. The reverse situation is more of a problem. Valine will form all of the contacts to the enzyme that isoleucine can form except for those to the missing methyl group. How much specificity could the absence of these contacts provide? Estimates of the differences in binding energy predict about a 200-fold discrimination, but since the actual error rate is found to be much lower, something in addition to a simple discrimination based on one binding reaction must contribute to specificity. An addi-tional step in the overall reaction in the form of editing by the synthetase increases the accuracy.

Although isoleucyl-tRNA synthetase can form a valyl adenylate com-plex, upon the addition of tRNA^Ile the tRNA is activated and then the

complex is immediately hydrolyzed. One way to think of this process is that activation is a two-step sieving process (Fig. 7.4). It permits the correct amino acid and similar but smaller amino acids to be activated. Then all amino acids smaller than the correct amino acid have a hydrolytic pathway available for removal of the misacylated amino acid. DNA synthesis and DNA cutting by restriction enzymes also use two-step error checking to achieve high accuracy. In the case of protein synthesis, fidelity is increased by identifying the amino acid several times, and for the DNA cutting enzymes, the nucleotide sequence is read more than once.