Two Residues in the Anticodon Recognition Domain of the Aspartyl-tRNA Synthetase from Pseudomonas aeruginosa Are Individually Implicated in the Recognition of tRNA Asn

Abstract

In many organisms, the formation of asparaginyl-tRNA is not done by direct aminoacylation of tRNA ^Asn but by specific tRNA-dependent transamidation of aspartyl-tRNA ^Asn . This transamidation pathway involves a nondiscriminating aspartyl-tRNA synthetase (AspRS) that charges both tRNA ^Asp and tRNA ^Asn with aspartic acid. Recently, it has been shown for the first time in an organism ( Pseudomonas aeruginosa PAO1) that the transamidation pathway is the only route of synthesis of Asn-tRNA ^Asn but does not participate in Gln-tRNA ^Gln formation. P. aeruginosa PAO1 has a nondiscriminating AspRS. We report here the identification of two residues in the anticodon recognition domain (H31 and G83) which are implicated in the recognition of tRNA ^Asn . Sequence comparisons of putative discriminating and nondiscriminating AspRSs (based on the presence or absence of the AdT operon and of AsnRS) revealed that bacterial nondiscriminating AspRSs possess a histidine at position 31 and usually a glycine at position 83, whereas discriminating AspRSs possess a leucine at position 31 and a residue other than a glycine at position 83. Mutagenesis of these residues of P. aeruginosa AspRS from histidine to leucine and from glycine to lysine increased the specificity of tRNA ^Asp charging over that of tRNA ^Asn by 3.5-fold and 4.2-fold, respectively. Thus, we show these residues to be determinants of the relaxed specificity of this nondiscriminating AspRS. Using available crystallographic data, we found that the H31 residue could interact with the central bases of the anticodons of the tRNA ^Asp and tRNA ^Asn . Therefore, these two determinants of specificity of P. aeruginosa AspRS could be important for all bacterial AspRSs.