Aminoacyl-tRNA synthetases (aaRSs) are multidomain proteins that specifically attach amino acids to their cognate tRNAs. Their most conserved, and presumably evolutionarily oldest, domains are the catalytic cores, which activate amino acids and transfer them to the 3 ends of tRNAs. Additional domains appended to or inserted in the body of aaRSs increase efficiency and specificity of the aminoacylation process, either by providing additional tRNA contacts, or by hydrolyzing noncognate amino acid products (cisediting). Here, we report specific tRNA-dependent trans-editing by aaRS-like proteins that reciprocate the editing domains of aaRSs, but not the remainder of the corresponding enzyme. A freestanding homologue of the prolyl-tRNA synthetase-editing domain, the PrdX protein from Clostridium sticklandii, efficiently and specifically hydrolyzes Ala-tRNA Pro . Similarly, autonomous alanyl-tRNA synthetase-editing domain homologues (AlaX proteins) from Methanosarcina barkeri and Sulfolobus solfataricus hydrolyze SertRNA Ala and Gly-tRNA Ala substrates. The discovery of autonomous editing proteins efficient in hydrolyzing misacylated products provides a direct link between ancestral aaRSs consisting solely of the catalytic core and extant enzymes to which functionally independent modules are appended.

Threonyl-tRNA synthetase (ThrRS) participates in protein synthesis quality control by selectively editing the misacylated species SertRNA Thr . In bacteria and eukaryotes the editing function of ThrRS resides in a highly conserved N-terminal domain distant from the active site. Most archaeal ThrRS proteins are devoid of this editing domain, suggesting evolutionary divergence of quality-control mechanisms. Here we show that archaeal editing of Ser-tRNA Thr is catalyzed by a domain unrelated to, and absent from, bacterial and eukaryotic ThrRSs. Despite the lack of sequence homology, the archaeal and bacterial editing domains are both reliant on a pair of essential histidine residues suggestive of a common catalytic mechanism. Whereas the archaeal editing module is most commonly part of full-length ThrRS, several crenarchaeal species contain individual genes encoding the catalytic (ThrRS-cat) and editing domains (ThrRS-ed). Sulfolobus solfataricus ThrRS-cat was shown to synthesize both Thr-tRNA Thr and Ser-tRNA Thr and to lack editing activity against Ser-tRNA Thr . In contrast, ThrRS-ed lacks aminoacylation activity but can act as an autonomous protein in trans to hydrolyze specifically Ser-tRNA Thr , or it can be fused to ThrRS-cat to provide the same function in cis. Deletion analyses indicate that ThrRS-ed is dispensable for growth of S. solfataricus under standard conditions but is required for normal growth in media with elevated serine levels. The growth phenotype of the ThrRS-ed deletion strain suggests that retention of the discontinuous ThrRS quaternary structure relates to specific physiological requirements still evident in certain Archaea.

Two dissimilar seryl-transfer RNA (tRNA) synthetases (SerRSs) exist in Methanosarcina barkeri, one of bacterial type and the other resembling SerRSs present only in some methanogenic archaea. To investigate the requirements of these enzymes for tRNA Ser recognition, serylation of variant transcripts of M. barkeri tRNA Ser was kinetically analyzed in vitro with pure enzyme preparations. Characteristically for the serine system, the length of the variable arm was shown to be crucial for both enzymes, as was the identity of the discriminator base (G73). Moreover, a novel determinant for the specific tRNA Ser recognition was identified as the anticodon stem base pair G30:C40; its contribution to the efficiency of serylation was remarkable for both SerRSs. However, despite these similarities, the two SerRSs do not possess a uniform mode of tRNA Ser recognition, and additional determinants are necessary for serylation specificity by the methanogenic enzyme. In particular, the methanogenic SerRS relies on G1:C72 identity and on the number of unpaired nucleotides at the base of the variable stem for tRNA Ser recognition, unlike its bacterial type counterpart. We propose that such a distinction between the two enzymes in tRNA Ser identity determinants reflects their evolutionary pathways, hence attesting to their diversity.To maintain translational accuracy, aminoacyl-transfer RNA (tRNA) 1 synthetases are highly selective toward their amino acid and tRNA substrates. In the process of tRNA recognition, the cognate and non-cognate substrates are discriminated according to characteristic nucleotides in certain positions of the tRNA, specific for the tRNA/synthetase system.