Recognizing the D-loop of transfer RNAs.

M that maintain fidelity and repair mistakes are ubiquitous throughout the protein biosynthesis pathway (1). The aminoacyl tRNAs serve as critical turning points in translation, because they link the nucleic acid genetic code with the amino acid building blocks of proteins. Misacylation of tRNAs can have devastating results, affecting the very survivability of an organism. Accuracy in tRNA aminoacylation therefore is paramount to the fidelity of the genetic code. Aminoacyl tRNAs are generated typically through action of the diverse family of aminoacyl-tRNA synthetases (AARSs). One of the hallmarks of these enzymes is the exquisite specificity with which each selects and aminoacylates only its cognate tRNA(s) with only its cognate amino acid (2–5). Herculean efforts spanning nearly 25 years have produced crystal structures for 19 of the 20 AARSs (alanyl-tRNA synthetase is the last holdout). Many of these structures provide detailed molecular insight into the nature of tRNA recognition and discrimination by either providing a structure of the enzyme in complex with its cognate tRNA or suggesting a model that can be tested biochemically. Thus, a clear picture is beginning to emerge, delineating some of the common and not-so-common themes used by the AARSs to discriminate cognate from noncognate tRNAs. [There are several reviews available that describe tRNA recognition by AARSs in detail (4–7).] Each AARS recognizes specific identity elements within its cognate tRNA(s); these nucleotides are often rigorously conserved and are typically clustered in the anticodon stem loop and the acceptor stem of the tRNA (Fig. 1). [For a comprehensive review on tRNA identity, see Giegé et al. (4).] The crystal structures of several different tRNA AARS complexes demonstrate that most AARSs form specific hydrogen bonding arrays with identity elements in either or both of these tRNA regions (Fig. 1 A) and occasionally with the variable arm (Fig. 1, VA) as well. In contrast, the rest of a given tRNA is often neglected by its AARS, with the junction of the D-loop and T C-loop extending away from the enzyme (for an example, see Fig. 2B). On page 13537 of this issue of PNAS, Shimada et al. (8) present a detailed structural analysis of ArgRS from T. thermophilus and suggest a mechanism by which this enzyme recognizes an unusual D-loop identity element in tRNAArg (A20). Nucleotide A20 is conserved in most known tRNAArg isoacceptors and has been identified as an identity element both in vitro and in vivo (9–11). Exceptions include S. cerevisiae, Neurospora crassa, Schizosaccharomyces pombe, and the mitochondria of animals and single cell eukaryotes, where position 20 is less conserved and can be U, C, or D (dihydrouridine; ref. 12). A cocrystal structure of S. cerevisiae ArgRS with a tRNAArg isoacceptor containing D20 has been reported previously (13). In this crystal structure, D20 is recognized by Asn106, Phe109, and Gln111 (Fig. 1B, Phe109 stacks against the D20 aromatic ring and is not shown). On the basis of structural similarities between the T. thermophilus ArgRS structure and the published S. cerevisiae ArgRS tRNAArg complex (13), Shimada et al. (8) were able to dock T. thermophilus tRNAArg (containing A20) onto their structure of T. thermophilus ArgRS (Fig. 2A). In this model, Asn106 of S. cerevisiae ArgRS is replaced by Val74 in T. thermophilus ArgRS. The smaller Val74 side chain creates a wider cavity to accommodate the larger A20 nucleotide. In contrast, Tyr77 and Asn79 (the two residues that align with Phe109 and Gln111 of S. cerevisiae ArgRS) are proximal to A20 but are too far removed to form direct contacts. This observation led the authors to propose and biochemically evaluate possible local rearrangements in the T. thermophilus ArgRS tRNAArg complex. Site-directed mutagenesis of Asn79 in ArgRS and or A20 in tRNAArg, followed by kinetic analyses of each new tRNAArg ArgRS combination, demonstrated that Asn79 indeed is involved directly in recognition of A20. A local structural model was constructed to reflect the proposed conformational reorganization after tRNAArg binding. In this final model, A20 directly contacts the Asn79 carboxyamide side chain via two hydrogen bonds [Figs. 1C and 4C in the accompanying paper (8)]. The results of these two crystallographic and biochemical analyses of ArgRS reveal a detailed molecular picture of the two different mechanisms used by this enzyme to recognize a D-loop identity element in tRNAArg (compare Fig. 1 B and C; refs. 8 and 13).