Structural and mutational studies of the recognition of the arginine tRNA-specific major identity element, A20, by arginyl-tRNA synthetase

Arginyl-tRNA synthetase (ArgRS) recognizes two major identity elements of tRNA^Arg: A20, located at the outside corner of the L-shaped tRNA, and C35, the second letter of the anticodon. Only a few exceptional organisms, such as the yeast Saccharomyces cerevisiae, lack A20 in tRNA^Arg. In the present study, we solved the crystal structure of a typical A20-recognizing ArgRS from Thermus thermophilus at 2.3 Å resolution. The structure of the T. thermophilus ArgRS was found to be similar to that of the previously reported S. cerevisiae ArgRS, except for short insertions and a concomitant conformational change in the N-terminal domain. The structure of the yeast ArgRS-tRNA^Arg complex suggested that two residues in the unique N-terminal domain, Tyr⁷⁷ and Asn⁷⁹, which are phylogenetically invariant in the ArgRSs from all organisms with A20 in tRNA^Args, are involved in A20 recognition. However, in a docking model constructed based on the yeast ArgRS-tRNA^Arg and T. thermophilus ArgRS structures, Tyr⁷⁷ and Asn⁷⁹ are not close enough to make direct contact with A20, because of the conformational change in the N-terminal domain. Nevertheless, the replacement of Tyr⁷⁷ or Asn⁷⁹ by Ala severely reduced the arginylation efficiency. Therefore, some conformational change around A20 is necessary for the recognition. Surprisingly, the N79D mutant equally recognized A20 and G20, with only a slight reduction in the arginylation efficiency as compared with the wild-type enzyme. Other mutants of Asn⁷⁹ also exhibited broader specificity for the nucleotide at position 20 of tRNA^Arg. We propose a model of A20 recognition by the ArgRS that is consistent with the present results of the mutational analyses.