Turning tRNA upside down: When aminoacylation is not a prerequisite to protein synthesis.

I n the first 20 years since their discovery, the aminoacyl-tRNA synthetases (aaRS) were considered purely for their roles in protein synthesis. Each aaRS links its cognate amino acid to one of the tRNAs bearing the appropriate anticodon trinucleotides before decoding and translation by the ribosome. The accuracy of this aminoacylation reaction depends on the ability of each aaRS to select its cognate substrates from pools of chemically similar amino acid and tRNA substrates, which in turn relies on the presence of highly specific binding sites for both nucleic acids and amino acids. In the original formulation of Crick’s adaptor hypothesis, each amino acid and corresponding tRNA was envisioned to match a single cognate aaRS, but this view has largely been overturned (1). Large-scale sequencing efforts revealed that many genomes of extant organisms contain both duplications of full-length tRNA synthetases, as well as aaRS-like proteins, which are evolutionarily conserved fragments reiterating functional domains from many known synthetases (2, 3). Functions have been deduced for only a small fraction of these proteins, and, in most cases, it is the catalytic core domains that have been recruited for a variety of new roles. These include roles in amino acid biosynthesis (4, 5), DNA replication processivity (6), and protein synthesis quality control (7, 8). The paper by Salazar et al. (9) in this issue of PNAS adds to this list by describing how the glutamyl-tRNA synthetase (GluRS) paralog YadB attaches glutamate to queuosine, generating a hypermodified nucleoside at the first anticodon position of tRNAAsp. This surprising finding provides a further dramatic illustration of the catalytic and RNA recognition versatility of the class I aaRS catalytic fold, and it implies that tRNA modifications might be more widespread than previously anticipated, owing to their chemical lability. The function of YadB, which lacks the C-terminal anticodon-binding domain of GluRS, had been the subject of speculation for some time. YadB was first observed in the emerging Escherichia coli genome but was later found to be relatively widespread in bacterial genomes. Initially, YadB was proposed to be a relic of an ancient transamidation pathway (10). That particular hypothesis postulated that the ancestor of YadB was a GluRS able to attach glutamate to the noncognate substrate tRNAGln. Recent studies confirmed YadB as a structural analogue of the catalytic core of GluRS that has retained the ability to activate glutamate (11), albeit without the need for tRNA binding that characterizes GluRS (12). Closer examination revealed that YadB does not attach activated glutamate to either tRNAGln or tRNAGlu (11), but instead to tRNAAsp (9, 13). Although the latter report by Dubois et al. (13) in this issue of PNAS provided unequivocal evidence for the surprising aminoacylation of tRNAAsp, it left unanswered the more vexing question of what function, if any, this apparent heterologous aminoacylation serves in vivo. The surprising answer to the question of YadB’s function is provided by the work of Salazar et al. (9). In addition to confirming what was reported concerning YadB (11, 13), Salazar et al. establish a truly surprising fate for the amino acid in YadB aminoacylation: rather than being transferred to the 3 end of the tRNA, the glutamate becomes attached to the hypermodified nucleoside queuosine (Q) at the first anticodon position, leading to the formation of glutamyl-Q (Fig. 1). They base this remarkable conclusion on three telling observations. First, whereas a typical 3 aminoacylated tRNA is resistant to treatment with periodate, the YadB aminoacylation product exhibits the same decrease in length as unacylated tRNA upon periodate treatment. Second, use of aspartyl-tRNA synthetase (AspRS) and YadB in concert with pure tRNAAsp substrate produces a product that can be labeled with both radioactive glutamate and aspartate. Third, mass spectroscopy analysis of tRNA hydrolysates provides definitive physical evidence that the glutamate is transferred to queuosine. Because the only position in tRNAAsp that exhibits the Q modification is the wobble base of the anticodon (nucleotide 34), the glutamate modification is restricted to a single nucleotide. Consistent with this hypothesis, YadB is unable to aminoacylate transcripts of tRNAAsp, and tRNA prepared from an E. coli strain deficient in queuosine synthesis is refractory to modification. Consequently, YadB provides one of the first definitive cases of a ‘‘closely