Aminoacyl-tRNAs are substrates for translation and are pivotal in determining how the genetic code is interpreted as amino acids. The function of aminoacyl-tRNA synthesis is to precisely match amino acids with tRNAs containing the corresponding anticodon. This is primarily achieved by the direct attachment of an amino acid to the corresponding tRNA by an aminoacyl-tRNA synthetase, although intrinsic proofreading and extrinsic editing are also essential in several cases. Recent studies of aminoacyl-tRNA synthesis, mainly prompted by the advent of whole genome sequencing and the availability of a vast body of structural data, have led to an expanded and more detailed picture of how aminoacyl-tRNAs are synthesized. This article reviews current knowledge of the biochemical, structural, and evolutionary facets of aminoacyl-tRNA synthesis.

SUMMARY The aminoacyl-tRNA synthetases (AARSs) and their relationship to the genetic code are examined from the evolutionary perspective. Despite a loose correlation between codon assignments and AARS evolutionary relationships, the code is far too highly structured to have been ordered merely through the evolutionary wanderings of these enzymes. Nevertheless, the AARSs are very informative about the evolutionary process. Examination of the phylogenetic trees for each of the AARSs reveals the following. (i) Their evolutionary relationships mostly conform to established organismal phylogeny: a strong distinction exists between bacterial- and archaeal-type AARSs. (ii) Although the evolutionary profiles of the individual AARSs might be expected to be similar in general respects, they are not. It is argued that these differences in profiles reflect the stages in the evolutionary process when the taxonomic distributions of the individual AARSs became fixed, not the nature of the individual enzymes. (iii) Horizontal transfer of AARS genes between Bacteria and Archaea is asymmetric: transfer of archaeal AARSs to the Bacteria is more prevalent than the reverse, which is seen only for the “gemini group.” (iv) The most far-ranging transfers of AARS genes have tended to occur in the distant evolutionary past, before or during formation of the primary organismal domains. These findings are also used to refine the theory that at the evolutionary stage represented by the root of the universal phylogenetic tree, cells were far more primitive than their modern counterparts and thus exchanged genetic material in far less restricted ways, in effect evolving in a communal sense.

Translating the 4-letter code of RNA into the 22-letter alphabet of proteins is a central feature of cellular life. The fidelity with which mRNA is translated during protein synthesis is determined by two factors: the availability of aminoacyl-tRNAs composed of cognate amino acid:tRNA pairs and the accurate selection of aminoacyl-tRNAs on the ribosome. The role of aminoacyl-tRNA synthetases in translation is to define the genetic code by accurately pairing cognate tRNAs with their corresponding amino acids. Synthetases achieve the amino acid substrate specificity necessary to keep errors in translation to an acceptable level in two ways: preferential binding of the cognate amino acid and selective editing of near-cognate amino acids. Editing significantly decreases the frequency of errors and is important for translational quality control, and many details of the various editing mechanisms and their effect on different cellular systems are now starting to emerge.

Several methanogenic archaea lack cysteinyl-transfer RNA (tRNA) synthetase (CysRS), the essential enzyme that provides Cys-tRNA(Cys) for translation in most organisms. Partial purification of the corresponding activity from Methanocaldococcus jannaschii indicated that tRNA(Cys) becomes acylated with O-phosphoserine (Sep) but not with cysteine. Further analyses identified a class II-type O-phosphoseryl-tRNA synthetase (SepRS) and Sep-tRNA:Cys-tRNA synthase (SepCysS). SepRS specifically forms Sep-tRNA(Cys), which is then converted to Cys-tRNA(Cys) by SepCysS. Comparative genomic analyses suggest that this pathway, encoded in all organisms lacking CysRS, can also act as the sole route for cysteine biosynthesis. This was proven for Methanococcus maripaludis, where deletion of the SepRS-encoding gene resulted in cysteine auxotrophy. As the conversions of Sep-tRNA to Cys-tRNA or to selenocysteinyl-tRNA are chemically analogous, the catalytic activity of SepCysS provides a means by which both cysteine and selenocysteine may have originally been added to the genetic code.

Transfer RNAs (tRNA) are best known for their role as adaptors during translation of the genetic code. Beyond their canonical role during protein biosynthesis, tRNAs also perform additional functions in both prokaryotes and eukaryotes for example in regulating gene expression. Aminoacylated tRNAs have also been implicated as substrates for non-ribosomal peptide bond formation, post-translational protein labeling, modification of phospholipids in the cell membrane, and antibiotic biosyntheses. Most recently tRNA fragments, or tRFs, have also been recognized to play regulatory roles. Here, we examine in more detail some of the new functions emerging for tRNA in a variety of cellular processes outside of protein synthesis.

SUMMARY We report an interaction between poxA, encoding a paralog of lysyl tRNA-synthetase, and the closely linked yjeK gene, encoding a putative 2,3-β-lysine aminomutase, that is critical for virulence and stress resistance in Salmonella enterica. Salmonella poxA and yjeK mutants share extensive phenotypic pleiotropy including attenuated virulence in mice, an increased ability to respire under nutrient limiting conditions, hypersusceptibility to a variety of diverse growth inhibitors and altered expression of multiple proteins including several encoded on the SPI-1 pathogenicity island. PoxA mediates post-translational modification of bacterial elongation factor P (EF-P), analogous to the modification of the eukaryotic EF-P homologue, eIF5A, with hypusine. The modification of EF-P is a mechanism of regulation whereby PoxA acts as an aminoacyl-tRNA synthetase that attaches an amino acid to a protein resembling tRNA rather than to a tRNA.

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.

The aminoacyl-tRNA synthetases are an essential and universally distributed family of enzymes that plays a critical role in protein synthesis, pairing tRNAs with their cognate amino acids for decoding mRNAs according to the genetic code. Synthetases help to ensure accurate translation of the genetic code by using both highly accurate cognate substrate recognition and stringent proofreading of noncognate products. While alterations in the quality control mechanisms of synthetases are generally detrimental to cellular viability, recent studies suggest that in some instances such changes facilitate adaption to stress conditions. Beyond their central role in translation, synthetases are also emerging as key players in an increasing number of other cellular processes, with far-reaching consequences in health and disease. The biochemical versatility of the synthetases has also proven pivotal in efforts to expand the genetic code, further emphasizing the wide-ranging roles of the aminoacyl-tRNA synthetase family in synthetic and natural biology.