Two residues in the anticodon recognition domain of the aspartyl-tRNA synthetase from Pseudomonas aeruginosa are individually implicated in the recognition of tRNAAsn.
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.
Bernard
Dominic
D
Centre de Recherche en Infectiologie, CHU Laval, 2705 Boulevard Laurier, RC-709, Sainte-Foy, Quebec, Canada G1V 4G2.
Akochy
Pierre-Marie
PM
Beaulieu
David
D
Lapointe
Jacques
J
Roy
Paul H
PH
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000926:Anticodon; D001226:Aspartate-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015964:Gene Expression Regulation, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D012348:RNA, Transfer, Asn / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
D000926:Anticodon; D012348:RNA, Transfer, Asn; D001226:Aspartate-tRNA Ligase