Aminoacyl-transfer RNA (tRNA) synthetases, which catalyze the attachment of the correct amino acid to its corresponding tRNA during translation of the genetic code, are proven antimicrobial drug targets. We show that the broad-spectrum antifungal 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), in development for the treatment of onychomycosis, inhibits yeast cytoplasmic leucyl-tRNA synthetase by formation of a stable tRNA(Leu)-AN2690 adduct in the editing site of the enzyme. Adduct formation is mediated through the boron atom of AN2690 and the 2'- and 3'-oxygen atoms of tRNA's3'-terminal adenosine. The trapping of enzyme-bound tRNA(Leu) in the editing site prevents catalytic turnover, thus inhibiting synthesis of leucyl-tRNA(Leu) and consequentially blocking protein synthesis. This result establishes the editing site as a bona fide target for aminoacyl-tRNA synthetase inhibitors.

The crystal structure of Thermus thermophilus seryl-transfer RNA synthetase, a class 2 aminoacyl-tRNA synthetase, complexed with a single tRNA(Ser) molecule was solved at 2.9 A resolution. The structure revealed how insertion of conserved base G20b from the D loop into the core of the tRNA determines the orientation of the long variable arm, which is a characteristic feature of most serine specific tRNAs. On tRNA binding, the antiparallel coiled-coil domain of one subunit of the synthetase makes contacts with the variable arm and T psi C loop of the tRNA and directs the acceptor stem of the tRNA into the active site of the other subunit. Specificity depends principally on recognition of the shape of tRNA(Ser) through backbone contacts and secondarily on sequence specific interactions.

The aminoacyl-tRNA synthetases link tRNAs with their cognate amino acid. In some cases, their fidelity relies on hydrolytic editing that destroys incorrectly activated amino acids or mischarged tRNAs. We present structures of leucyl-tRNA synthetase complexed with analogs of the distinct pre- and posttransfer editing substrates. The editing active site binds the two different substrates using a single amino acid discriminatory pocket while preserving the same mode of adenine recognition. This suggests a similar mechanism of hydrolysis for both editing substrates that depends on a key, completely conserved aspartic acid, which interacts with the alpha-amino group of the noncognate amino acid and positions both substrates for hydrolysis. Our results demonstrate the economy by which a single active site accommodates two distinct substrates in a proofreading process critical to the fidelity of protein synthesis.

Leucyl-, isoleucyl-and valyl-tRNA synthetases are closely related large monomeric class I synthetases. Each contains a homologous insertion domain of~200 residues, which is thought to permit them to hydrolyse (`edit') cognate tRNA that has been mischarged with a chemically similar but non-cognate amino acid. We describe the ®rst crystal structure of a leucyl-tRNA synthetase, from the hyperthermophile Thermus thermophilus, at 2.0 A Ê resolution. The overall architecture is similar to that of isoleucyl-tRNA synthetase, except that the putative editing domain is inserted at a different position in the primary structure. This feature is unique to prokaryote-like leucyl-tRNA synthetases, as is the presence of a novel additional exibly inserted domain. Comparison of native enzyme and complexes with leucine and a leucyladenylate analogue shows that binding of the adenosine moiety of leucyl-adenylate causes signi®cant conformational changes in the active site required for amino acid activation and tight binding of the adenylate. These changes are propagated to more distant regions of the enzyme, leading to a signi®cantly more ordered structure ready for the subsequent aminoacylation and/or editing steps.

Leucyl-tRNA synthetase (LeuRS) has a specific post-transfer editing activity directed against mischarged isoleucine and similar noncognate amino acids. We describe the post-transfer-editing and product complexes of Thermus thermophilus LeuRS (LeuRSTT) with tRNA(Leu) at 2.9- to 3.3-A resolution. In the post-transfer-editing configuration, A76 binds in the editing active site exactly as previously found for the adenosine moiety of a small-molecule editing-substrate analog. The 60 C-terminal residues of LeuRSTT, unseen in previous structures, fold into a compact domain flexibly linked to the rest of the molecule and interacting with the G19-C56 tertiary base pair of tRNA(Leu). LeuRS recognition of tRNA(Leu) depends essentially on tRNA shape rather than base-specific interactions. The structures show that considerable domain rotations, notably of the editing domain, accompany the tRNA-3' end dynamics associated successively with aminoacylation, post-transfer editing and product release.

Bacterial tyrosyl-tRNA synthetases (TyrRS) possess ā exibly linked C-terminal domain of~80 residues, which has hitherto been disordered in crystal structures of the enzyme. We have determined the structure of Thermus thermophilus TyrRS at 2.0 A Ê resolution in a crystal form in which the C-terminal domain is ordered, and con®rm that the fold is similar to part of the C-terminal domain of ribosomal protein S4. We have also determined the structure at 2.9 A Ê resolution of the complex of T.thermophilus TyrRS with cognate tRNA tyr (GYA). In this structure, the C-terminal domain binds between the characteristic long variable arm of the tRNA and the anti-codon stem, thus recognizing the unique shape of the tRNA. The anticodon bases have a novel conformation with A-36 stacked on G-34, and both G-34 and Y-35 are base-speci®cally recognized. The tRNA binds across the two subunits of the dimeric enzyme and, remarkably, the mode of recognition of the class I TyrRS for its cognate tRNA resembles that of a class II synthetase in being from the major groove side of the acceptor stem. Keywords: class I aminoacyl-tRNA synthetase/ribosomal protein S4/tRNA recognition/tyrosyl-tRNA synthetase/ X-ray crystallography IntroductionTyrosyl-tRNA synthetase (TyrRS) is a class I aminoacyltRNA synthetase, but is unusual in that it is a functional dimer, a feature only shared with tryptophanyl-tRNA synthetase amongst class I synthetases (Cusack, 1995). It was the ®rst synthetase to have its crystal structure solved (Bhat et al., 1982), including its substrate complexes with tyrosine and tyrosyl-adenylate (Brick and Blow, 1987;Brick et al., 1989), and both the mechanism of tyrosyladenylate formation (reviewed in Fersht, 1987;First, 1997) and its interaction with cognate tRNA tyr (reviewed in Bedouelle, 1990;Bedouelle et al., 1993) have been the subject of intense biochemical study. However, there remain a number of signi®cant areas where structural data that correlate with biochemical observations are lacking. Here we report the ®rst crystal structure of a bacterial tyrosyl-tRNA synthetase complexed with cognate tRNA tyr . This permits us to visualize the mode of interaction of this synthetase for its cognate tRNA, which in prokaryotes, but not archaea or eukaryotes, is of the class 2 type, i.e. with a long variable arm. The enzyme subunit comprises an N-terminal Rossmann-fold catalytic domain, characteristic of class I synthetases, followed by a central a-helical domain and, ®nally, a putative tRNAbinding C-terminal domain of~80 residues, which has been invisible until now due to disorder in all crystal structures of TyrRS (Brick et al., 1989;Qiu et al., 2001). The structure described here reveals the exact role in speci®c tRNA recognition of the¯exibly linked C-terminal domain and also shows how unique tertiary interactions in the core of tRNA tyr lead to a different orientation of the long variable arm, permitting discrimination from other long variable arm (class 2) tRNAs such as tRNA ser . The structure con®rms the expected cross-subun...

The archaeal/eukaryotic tyrosyl-tRNA synthetase (TyrRS)-tRNA(Tyr) pairs do not cross-react with their bacterial counterparts. This 'orthogonal' condition is essential for using the archaeal pair to expand the bacterial genetic code. In this study, the structure of the Methanococcus jannaschii TyrRS-tRNA(Tyr)-L-tyrosine complex, solved at a resolution of 1.95 A, reveals that this archaeal TyrRS strictly recognizes the C1-G72 base pair, whereas the bacterial TyrRS recognizes the G1-C72 in a different manner using different residues. These diverse tRNA recognition modes form the basis for the orthogonality. The common tRNA(Tyr) identity determinants (the discriminator, A73 and the anticodon residues) are also recognized in manners different from those of the bacterial TyrRS. Based on this finding, we created a mutant TyrRS that aminoacylates the amber suppressor tRNA with C34 65 times more efficiently than does the wild-type enzyme.

The low temperature crystal structure of the ternary complex of Thermus thermophilus seryl‐tRNA synthetase with tRNA(Ser) (GGA) and a non‐hydrolysable seryl‐adenylate analogue has been refined at 2.7 angstrom resolution. The analogue is found in both active sites of the synthetase dimer but there is only one tRNA bound across the two subunits. The motif 2 loop of the active site into which the single tRNA enters interacts within the major groove of the acceptor stem. In particular, a novel ring‐ring interaction between Phe262 on the extremity of this loop and the edges of bases U68 and C69 explains the conservation of pyrimidine bases at these positions in serine isoaccepting tRNAs. This active site takes on a significantly different ordered conformation from that observed in the other subunit, which lacks tRNA. Upon tRNA binding, a number of active site residues previously found interacting with the ATP or adenylate now switch to participate in tRNA recognition. These results shed further light on the structural dynamics of the overall aminoacylation reaction in class II synthetases by revealing a mechanism which may promote an ordered passage through the activation and transfer steps.