EVIDENCE FOR THE DOUBLE-SIEVE EDITING MECHANISM IN PROTEIN-SYNTHESIS - STERIC EXCLUSION OF ISOLEUCINE BY VALYL-TRANSFER RNA-SYNTHETASES

Evidence is presented for a simple stereochemical model, the “double sieve”, by which the exquisite fidelity of the genetic coding process is preserved at the level of charging tRNA with the correct amino acid. The high accuracy in the recognition of amino acids is achieved by the synthetic and hydrolytic (editing) sites on an aminoacyl-tRNA synthetase functioning as a pair of sieves, crudely sorting the amino acids according to size as well as chemical nature. The synthetic site rejects, at a tolerable level, amino acids larger than the specific substrate; the hydrolytic site destroys the reaction products of the amino acids which are smaller than (or isosteric with) the specific substrate. Testing hypothetical editing mechanisms by mapping out the range of misactivations catalyzed by the aminoacyl-tRNA synthetases is hampered by trace impurities of the specific amino acid in preparations of the nonspecific amino acids. An enzymic method is given for scavenging these impurities. It is found that at least 97% of the apparent isoleucine-dependent pyrophosphate exchange activity of three representative valyl-tRNA synthetases is attributable to residual traces of valine. The selectivity of the enzyme from Escherichia coli against isoleucine is greater than 6 × 104. Combined with the known ratio of concentrations of isoleucine and valine in vivo, an error rate of less than 3 × 10-6 is calculated for the mistaken activation of isoleucine for valine. Isoleucine is thus sieved out at the activation step and the rate of formation of Ile-tRNAVal is so low as not to require subsequent editing. © 1979, American Chemical Society. All rights reserved.