Evolutionary Divergence of the Archaeal Aspartyl-tRNA Synthetases into Discriminating and Nondiscriminating Forms

Abstract

Asparaginyl-tRNA (Asn-tRNA) is generated in nature via two alternate routes, either direct acylation of tRNA with asparagine by asparaginyl-tRNA synthetase (AsnRS) or in a two-step pathway that requires misacylated Asp-tRNA Asn as an intermediate. This misacylated aminoacyl-tRNA is formed by a nondiscriminating aspartyltRNA synthetase (AspRS), an enzyme that in addition to forming Asp-tRNA Asp also misacylates tRNA Asn . In contrast, a discriminating AspRS cannot acylate tRNA Asn . It has been suggested that the archaeal AspRS enzymes are nondiscriminating, whereas the bacterial ones discriminate. The archaeal and bacterial AspRS proteins are indeed distinct in sequence and structure. However, we show that both discriminating and nondiscriminating forms of AspRS exist among the archaea. Using unfractionated methanobacterial and pyrococcal tRNA, the Methanothermobacter thermautotrophicus AspRS acylated approximately twice as much tRNA as did AspRS from Pyrococcus kodakaraensis or Ferroplasma acidarmanus. Proof that Asp-tRNA Asn was generated by the methanogen synthetase was the conversion of AsptRNA formed by M. thermautotrophicus AspRS to AsntRNA by M. thermautotrophicus Asp-tRNA Asn amidotransferase. In contrast, Asp-tRNA formed by the Pyrococcus or Ferroplasma enzymes was not a substrate for the amidotransferase. Also, although all three AspRS enzymes charged tRNA Asp transcripts, only M. thermautotrophicus AspRS aspartylated the tRNA Asn transcript. Genomic analysis provides a rationale for the nature of these enzymes. The mischarging AspRS correlates with the absence in the genome of AsnRS and the presence of Asp-tRNA Asn amidotransferase, employed by the transamidation pathway. In contrast, the discriminating AspRS correlates with the absence of the amidotransferase and the presence of AsnRS, forming Asn-tRNA by direct aminoacylation. The high sequence identity, up to 60% between discriminating and nondiscriminating archaeal AspRSs, suggests that few mutational steps may be necessary to convert the tRNA-discriminating ability of a tRNA synthetase.

Expanding tRNA recognition of a tRNA synthetase by a single amino acid change

“…Unfractionated P. furiosus tRNA (20 M) was aminoacylated with 160 M unlabeled aspartate and 0.1 M T. kodakaraensis or M. thermautotrophicus AspRS at 55°C for 30 min as described (15), or with 160 M unlabeled asparagine and 1 M E. coli AsnRS at 37°C for 30 min in 50 mM Hepes-KOH, pH 7.0͞50 mM KCl͞10 mM MgCl 2 ͞4 mM ATP͞5 mM DTT. Duplicate reactions with the same amount of 14 C-labeled amino acid were run in parallel to determine the aminoacylation time course.…”

“…The archaeal genre comprises all archaeal AspRS enzymes, as well as one of two types of AspRSs found in the bacteria Thermus thermophilus (12), Deinococcus radiodurans (13), and Clostridium acetobutylicum. Whereas the archaeal AspRS enzymes were thought to be nondiscriminating (12,14), it is now known that in several archaea (including Thermococcus kodakaraensis), this enzyme is discriminating (15). In contrast to bacterial AspRS proteins, the tRNA specificity of the archaeal enzyme can be predicted from the whole genome content (15): a ND-AspRS always exists in the genome together with the archaeal Asp-tRNA Asn amidotransferase, whereas a D-AspRS accompanies an asparaginyl-tRNA synthetase (AsnRS).…”

“…Mutant AspRS genes were generated by overlap-extension PCR and were cloned into pET20b between NdeI and BamHI sites so as to yield native proteins after overexpression. Purification of the mutant AspRSs was the same as for the wild-type enzyme (15). The sequences of the cloned AspRS genes were verified from minipreparations of the cultures from which the AspRSs were purified.…”

“…Cloning and Purification of the AspRS Proteins. Cloning and purification of AspRS from T. kodakaraensis, previously known as Pyrococcus kodakaraensis (22), has been described (15). Mutant AspRS genes were generated by overlap-extension PCR and were cloned into pET20b between NdeI and BamHI sites so as to yield native proteins after overexpression.…”

“…All enzymes were Ͼ95% pure, as judged by SDS͞PAGE followed by Coomassie blue staining. Methanothermobacter thermautotrophicus AspRS was prepared as described (15 (15). For measurement of kinetic parameters, initial velocities of aminoacylation were determined from the average of duplicate sets of data at various tRNA concentrations.…”