title: A non-discriminating aspartyl-tRNA synthetase from Halobacterium salinarum.
abstract: The tRNA-dependent transamidation pathway is the essential route for Asn-tRNA(Asn) formation in organisms that lack an asparaginyl-tRNA synthetase. This pathway relies on a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS encoded by aspS), an enzyme with relaxed tRNA specificity, to form Asp-tRNA(Asn). The misacylated tRNA is then converted to Asn-tRNA(Asn) by the action of an Asp-tRNA(Asn) amidotransferase. Here we show that Asn-tRNA(Asn) formation in the extreme halophile Halobacterium salinarum also occurs by this transamidation mechanism, and we explore the property of the haloarchaeal AspRS to aspartylate tRNA(Asn) in vivo and in vitro. Transformation of the E. coli trpA34 strain with the H. salinarum aspS and tRNA(Asn) genes led to restoration of tryptophan prototrophy by missense suppression of the trpA34 mutant with heterologously in vivo formed Asp-tRNA(Asn). The haloarchaeal AspRS works well at low and high (0.1-3 M) salt concentrations but it is unable to use Escherichia coli tRNA as substrate. We show that mutations of two amino acids (H26 and P84) located in the AspRS anticodon binding domain limit the specificity of this nondiscriminating enzyme towards tRNA(Asn). Thus, as was observed in an archaeal discriminating AspRS and a bacterial ND-AspRS, amino acids in these positions influence the enzyme's tRNA selection.
authors.affiliation: Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Alexander Machado
authors.initials: AM
authors.lastname: Cardoso
authors.forename: Carla
authors.initials: C
authors.lastname: Polycarpo
authors.forename: Orlando Bonifácio
authors.initials: OB
authors.lastname: Martins
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
mesh_terms: D001226:Aspartate-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins; D019617:Halobacterium salinarum / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D019884:Nitrogenous Group Transferases; D012348:RNA, Transfer, Asn; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D012348:RNA, Transfer, Asn; C412016:Asp-tRNA(Asn) amidotransferase; D019884:Nitrogenous Group Transferases; D001226:Aspartate-tRNA Ligase