Misacylation of tRNALys with noncognate amino acids by lysyl-tRNA synthetase.

Lysyl-tRNA synthetase (LysRS), a class II enzyme whose major function is to provide Lys-tRNALys for protein synthesis, also catalyzes aminoacylation of tRNALys with arginine, threonine, methionine, leucine, alanine, serine, and cysteine. The limited selectivity in the tRNA aminoacylation reaction appears to be due to inefficient editing of some amino acids (Met, Leu, Cys, Ala, Thr) by a pre-transfer mechanism or the absence of editing of other amino acids (Arg and Ser). Purified Arg-tRNALys, Thr-tRNALys, and Met-tRNALys were essentially not deacylated by LysRS, indicating that the enzyme does not possess a post-transfer editing mechanism. However, LysRS possesses an efficient pre-transfer editing mechanism which prevents misacylation of tRNALys with ornithine. A novel feature of this editing reaction is that ornithine lactam is formed by the facile cyclization of ornithyl adenylate.     

A conserved threonine within Escherichia coli leucyl-tRNA synthetase prevents hydrolytic editing of leucyl-tRNALeu

Aminoacyl-tRNA synthetases ensure the fidelity of protein synthesis by accurately selecting and activating cognate amino acids for aminoacylation of the correct tRNA. Some tRNA synthetases have evolved an editing active site that is separate from the amino acid activation site providing two steps or "sieves" for amino acid selection. These two sieves rely on different strategies for amino acid recognition to significantly enhance the accuracy of aminoacylation. We have performed alanine scanning mutagenesis in a conserved threonine-rich region of the Escherichia coli leucyl-tRNA synthetase's CP1 domain that is hypothesized to contain a putative editing active site. Characterization of purified mutant proteins led to the identification of a single conserved threonine that prevents the cognate leucine amino acid from being hydrolyzed after aminoacylation of the tRNA. Mutation of this threonine to an alanine eliminates discrimination of the cognate amino acid in the editing active site. This provides a molecular example of an amino acid discrimination mechanism in the tRNA synthetase's editing active site.     

Transiently misacylated tRNA is a primer for editing of misactivated adenylates by class I aminoacyl-tRNA synthetases

The genetic code depends on amino acid fine structure discrimination by aminoacyl-tRNA synthetases. For isoleucyl- (IleRS) and valyl-tRNA synthetases (ValRS), reactions that hydrolyze misactivated noncognate amino acids help to achieve high accuracy in aminoacylation. Two editing pathways contribute to aminoacylation fidelity: pretransfer and post-transfer. In pretransfer editing, the misactivated amino acid is hydrolyzed as an aminoacyl adenylate, while in post-transfer editing a misacylated tRNA is deacylated. Both reactions are dependent on a tRNA cofactor and require translocation to a site located approximately 30 A from the site of amino acid activation. Using a series of 3'-end modified tRNAs that are deficient in either aminoacylation, deacylation, or both, total editing (the sum of pre- and post-transfer editing) was shown to require both aminoacylation and deacylation activities. These and additional results with IleRS are consistent with a post-transfer deacylation event initiating formation of an editing-active enzyme/tRNA complex. In this state, the primed complex processively edits misactivated valyl-adenylate via the pretransfer route. Thus, misacylated tRNA is an obligatory intermediate for editing by either pathway.     

The balance between pre- and post-transfer editing in tRNA synthetases

The fidelity of tRNA aminoacylation is dependent in part on amino acid editing mechanisms. A hydrolytic activity that clears mischarged tRNAs typically resides in an active site on the tRNA synthetase that is distinct from its synthetic aminoacylation active site. A second pre-transfer editing pathway that hydrolyzes the tRNA synthetase aminoacyl adenylate intermediate can also be activated. Pre- and post-transfer editing activities can co-exist within a single tRNA synthetase resulting in a redundancy of fidelity mechanisms. However, in most cases one pathway appears to dominate, but when compromised, the secondary pathway can be activated to suppress tRNA synthetase infidelities.     

Misacylation and editing by Escherichia coli valyl-tRNA synthetase: evidence for two tRNA binding sites.

Valyl-tRNA synthetase (ValRS) has difficulty discriminating between its cognate amino acid, valine, and structurally similar amino acids. To minimize translational errors, the enzyme catalyzes a tRNA-dependent editing reaction that prevents accumulation of misacylated tRNA(Val). Editing occurs with threonine, alanine, serine, and cysteine, as well as with several nonprotein amino acids. The 3'-end of tRNA plays a vital role in promoting the tRNA-dependent editing reaction. Valine tRNA having the universally conserved 3'-terminal adenosine replaced by any other nucleoside does not stimulate the editing activity of ValRS. As a result 3'-end tRNA(Val) mutants, particularly those with 3'-terminal pyrimidines, are stably misacylated with threonine, alanine, serine, and cysteine. Valyl-tRNA synthetase is unable to hydrolytically deacylate misacylated tRNA(Val) terminating in 3'-pyrimidines but does deacylate mischarged tRNA(Val) terminating in adenosine or guanosine. Evidently, a purine at position 76 of tRNA(Val) is essential for translational editing by ValRS. We also observe misacylation of wild-type and 3'-end mutants of tRNA(Val) with isoleucine. Valyl-tRNA synthetase does not edit wild-type tRNA(Val)(A76) mischarged with isoleucine, presumably because isoleucine is only poorly accommodated at the editing site of the enzyme. Misacylated mutant tRNAs as well as 3'-end-truncated tRNA(Val) are mixed noncompetitive inhibitors of the aminoacylation reaction, suggesting that ValRS, a monomeric enzyme, may bind more than one tRNA(Val) molecule. Gel-mobility-shift experiments to characterize the interaction of tRNA(Val) with the enzyme provide evidence for two tRNA binding sites on ValRS.     

Transfer RNA-mediated editing in threonyl-tRNA synthetase. The class II solution to the double discrimination problem

Threonyl-tRNA synthetase, a class II synthetase, uses a unique zinc ion to discriminate against the isosteric valine at the activation step. The crystal structure of the enzyme with an analog of seryl adenylate shows that the noncognate serine cannot be fully discriminated at that step. We show that hydrolysis of the incorrectly formed ser-tRNA(Thr) is performed at a specific site in the N-terminal domain of the enzyme. The present study suggests that both classes of synthetases use effectively the ability of the CCA end of tRNA to switch between a hairpin and a helical conformation for aminoacylation and editing. As a consequence, the editing mechanism of both classes of synthetases can be described as mirror images, as already seen for tRNA binding and amino acid activation.     

Transfer RNA determinants for translational editing by Escherichia coli valyl-tRNA synthetase

Valyl-tRNA synthetase (ValRS) has difficulty differentiating valine from structurally similar non-cognate amino acids, most prominently threonine. To minimize errors in aminoacylation and translation the enzyme catalyzes a proofreading (editing) reaction that is dependent on the presence of cognate tRNA^Val. Editing occurs at a site functionally distinct from the aminoacylation site of ValRS and previous results have shown that the 3′-terminus of tRNA^Val is recognized differently at the two sites. Here, we extend these studies by comparing the contribution of aminoacylation identity determinants to productive recognition of tRNA^Val at the aminoacylation and editing sites, and by probing tRNA^Val for editing determinants that are distinct from those required for aminoacylation. Mutational analysis of Escherichia coli tRNA^Val and identity switch experiments with non-cognate tRNAs reveal a direct relationship between the ability of a tRNA to be aminoacylated and its ability to stimulate the editing activity of ValRS. This suggests that at least a majority of editing by the enzyme entails prior charging of tRNA and that misacylated tRNA is a transient intermediate in the editing reaction.     

Editing by a tRNA synthetase: DNA aptamer-induced translocation and hydrolysis of a misactivated amino acid

Aminoacyl-tRNA synthetases establish the rules of the genetic code by aminoacylation reactions. Occasional activation of the wrong amino acid can lead to errors of protein synthesis. For isoleucyl-tRNA synthetase, these errors are reduced by tRNA-dependent hydrolytic editing reactions that occur at a site 25 A from the active site. These reactions require that the misactivated amino acid be translocated from the active site to the center for editing. One mechanism describes translocation as requiring the mischarging of tRNA followed by a conformational change in the tRNA that moves the amino acid from one site to the other. Here a specific DNA aptamer is investigated. The aptamer can stimulate amino acid-specific editing but cannot be aminoacylated. Although the aptamer could in principle stimulate hydrolysis of a misactivated amino acid by an idiosyncratic mechanism, the aptamer is shown here to induce translocation and hydrolysis of misactivated aminoacyl adenylate at the same site as that seen with the tRNA cofactor. Thus, translocation to the site for editing does not require joining of the amino acid to the nucleic acid. Further experiments demonstrated that aptamer-induced editing is sensitive to aptamer sequence and that the aptamer is directed to a site other than the active site or tRNA binding site of the enzyme.     

The Mechanism of Pre-transfer Editing in Yeast Mitochondrial Threonyl-tRNA Synthetase

Accurate translation of mRNA into protein is a fundamental biological process critical for maintaining normal cellular functions. To ensure translational fidelity, aminoacyl-tRNA synthetases (aaRSs) employ pre-transfer and post-transfer editing activities to hydrolyze misactivated and mischarged amino acids, respectively. Whereas post-transfer editing, which requires either a specialized domain in aaRS or a trans-protein factor, is well described, the mechanism of pre-transfer editing is less understood. Here, we show that yeast mitochondrial threonyl-tRNA synthetase (MST1), which lacks an editing domain, utilizes pre-transfer editing to discriminate against serine. MST1 misactivates serine and edits seryl adenylate (Ser-AMP) in a tRNA-independent manner. MST1 hydrolyzes 80% of misactivated Ser-AMP at a rate 4-fold higher than that for the cognate threonyl adenylate (Thr-AMP) while releasing 20% of Ser-AMP into the solution. To understand the mechanism of pre-transfer editing, we solved the crystal structure of MST1 complexed with an analog of Ser-AMP. The binding of the Ser-AMP analog to MST1 induces conformational changes in the aminoacylation active site, and it positions a potential hydrolytic water molecule more favorably for nucleophilic attack. In addition, inhibition results reveal that the Ser-AMP analog binds the active site 100-fold less tightly than the Thr-AMP analog. In conclusion, we propose that the plasticity of the aminoacylation site in MST1 allows binding of Ser-AMP and the appropriate positioning of the hydrolytic water molecule.     

Functional group recognition at the aminoacylation and editing sites of E. coli valyl-tRNA synthetase

To correct misactivation and misacylation errors, Escherichia coli valyl-tRNA synthetase (ValRS) catalyzes a tRNA(Val)-dependent editing reaction at a site distinct from its aminoacylation site. Here we examined the effects of replacing the conserved 3'-adenosine of tRNA(Val) with nucleoside analogs, to identify structural elements of the 3'-terminal nucleoside necessary for tRNA function at the aminoacylation and editing sites of ValRS. The results show that the exocyclic amino group (N6) is not essential: purine riboside-substituted tRNA(Val) is active in aminoacylation and in stimulating editing. Presence of an O6 substituent (guanosine, inosine, xanthosine) interferes with aminoacylation as well as posttransfer and total editing (pre- plus posttransfer editing). Because ValRS does not recognize substituents at the 6-position, these results suggest that an unprotonated N1, capable of acting as an H-bond acceptor, is an essential determinant for both the aminoacylation and editing reactions. Substituents at the 2-position of the purine ring, either a 2-amino group (2-aminopurine, 2,6-diaminopurine, guanosine, and 7-deazaguanosine) or a 2-keto group (xanthosine, isoguanosine), strongly inhibit both aminoacylation and editing. Although aminoacylation by ValRS is at the 2'-OH, substitution of the 3'-terminal adenosine of tRNA(Val) with 3'-deoxyadenosine reduces the efficiency of valine acceptance and of posttransfer editing, demonstrating that the 3'-terminal hydroxyl group contributes to tRNA recognition at both the aminoacylation and editing sites. Our results show a strong correlation between the amino acid accepting activity of tRNA and its ability to stimulate editing, suggesting misacylated tRNA is a transient intermediate in the editing reaction, and editing by ValRS requires a posttransfer step.     

Pre-transfer editing by class II prolyl-tRNA synthetase: role of aminoacylation active site in "selective release" of noncognate amino acids

Aminoacyl-tRNA synthetases catalyze the attachment of cognate amino acids to specific tRNA molecules. To prevent potential errors in protein synthesis caused by misactivation of noncognate amino acids, some synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated tRNAs (post-transfer editing). In the case of post-transfer editing, synthetases employ a separate editing domain that is distinct from the site of amino acid activation, and the mechanism is believed to involve shuttling of the flexible CCA-3' end of the tRNA from the synthetic active site to the site of hydrolysis. The mechanism of pre-transfer editing is less well understood, and in most cases, the exact site of pre-transfer editing has not been conclusively identified. Here, we probe the pre-transfer editing activity of class II prolyl-tRNA synthetases from five species representing all three kingdoms of life. To locate the site of pre-transfer editing, truncation mutants were constructed by deleting the insertion domain characteristic of bacterial prolyl-tRNA synthetase species, which is the site of post-transfer editing, or the N- or C-terminal extension domains of eukaryotic and archaeal enzymes. In addition, the pre-transfer editing mechanism of Escherichia coli prolyl-tRNA synthetase was probed in detail. These studies show that a separate editing domain is not required for pre-transfer editing by prolyl-tRNA synthetase. The aminoacylation active site plays a significant role in preserving the fidelity of translation by acting as a filter that selectively releases non-cognate adenylates into solution, while protecting the cognate adenylate from hydrolysis.     

Transfer RNA modulates the editing mechanism used by class II prolyl-tRNA synthetase

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. To prevent errors in protein synthesis, many synthetases have evolved editing pathways by which misactivated amino acids (pre-transfer editing) and misacylated tRNAs (post-transfer editing) are hydrolyzed. Previous studies have shown that class II prolyl-tRNA synthetase (ProRS) possesses both pre- and post-transfer editing functions against noncognate alanine. To assess the relative contributions of pre- and post-transfer editing, presented herein are kinetic studies of an Escherichia coli ProRS mutant in which post-transfer editing is selectively inactivated, effectively isolating the pre-transfer editing pathway. When post-transfer editing is abolished, substantial levels of alanine mischarging are observed under saturating amino acid conditions, indicating that pre-transfer editing alone cannot prevent the formation of Ala-tRNA Pro. Steady-state kinetic parameters for aminoacylation measured under these conditions reveal that the preference for proline over alanine is 2000-fold, which is well within the regime where editing is required. Simultaneous measurement of AMP and Ala-tRNA Pro formation in the presence of tRNA Pro suggested that misactivated alanine is efficiently transferred to tRNA to form the mischarged product. In the absence of tRNA, enzyme-catalyzed Ala-AMP hydrolysis is the dominant form of editing, with "selective release" of noncognate adenylate from the active site constituting a minor pathway. Studies with human and Methanococcus jannaschii ProRS, which lack a post-transfer editing domain, suggest that enzymatic pre-transfer editing occurs within the aminoacylation active site. Taken together, the results reported herein illustrate how both pre- and post-transfer editing pathways work in concert to ensure accurate aminoacylation by ProRS.     

The homotetrameric phosphoseryl-tRNA synthetase from Methanosarcina mazei exhibits half-of-the-sites activity

Synthesis of cysteinyl-tRNA(Cys) in methanogenic archaea proceeds by a two-step pathway in which tRNA(Cys) is first aminoacylated with phosphoserine by phosphoseryl-tRNA synthetase (SepRS). Characterization of SepRS from the mesophile Methanosarcina mazei by gel filtration and nondenaturing mass spectrometry shows that the native enzyme exists as an alpha4 tetramer when expressed at high levels in Escherichia coli. However, active site titrations monitored by ATP/PP(i) burst kinetics, together with analysis of tRNA binding stoichiometry by fluorescence spectroscopy, show that the tetrameric enzyme binds two tRNAs and that only two of the four chemically equivalent subunits catalyze formation of phosphoseryl adenylate. Therefore, the phenomenon of half-of-the-sites activity, previously described for synthesis of 1 mol of tyrosyl adenylate by the dimeric class I tyrosyl-tRNA synthetase, operates as well in this homotetrameric class II tRNA synthetase. Analysis of cognate and noncognate reactions by ATP/PP(i) and aminoacylation kinetics strongly suggests that SepRS is able to discriminate against the noncognate amino acids glutamate, serine, and phosphothreonine without the need for a separate hydrolytic editing site. tRNA(Cys) binding to SepRS also enhances the capacity of the enzyme to discriminate among amino acids, indicating the existence of functional connectivity between the tRNA and amino acid binding sites of the enzyme.     

An aminoacyl-tRNA synthetase with a defunct editing site

Many aminoacyl-tRNA synthetases (aaRSs) contain two active sites, a synthetic site catalyzing aminoacyl-adenylate formation and tRNA aminoacylation and a second editing or proofreading site that hydrolyzes misactivated adenylates or mischarged tRNAs. The combined activities of these two sites lead to rigorous accuracy in tRNA aminoacylation, and both activities are essential to LeuRS and other aaRSs. Here, we describe studies of the human mitochondrial (hs mt) LeuRS indicating that the two active sites of this enzyme have undergone functional changes that impact how accurate aminoacylation is achieved. The sequence of the hs mt LeuRS closely resembles a bacterial LeuRS overall but displays significant variability in regions of the editing site. Studies comparing Escherichia coli and hs mt LeuRS reveal that the proofreading activity of the mt enzyme is disrupted by these sequence changes, as significant levels of Ile-tRNA(Leu) are formed in the presence of high concentrations of the noncognate amino acid. Experiments monitoring deacylation of Ile-tRNA(Leu) and misactivated adenylate turnover revealed that the editing active site is not operational. However, hs mt LeuRS has weaker binding affinities for both cognate and noncognate amino acids relative to the E. coli enzyme and an elevated discrimination ratio. Therefore, the enzyme achieves fidelity using a more specific synthetic active site that is not prone to errors under physiological conditions. This enhanced specificity must compensate for the presence of a defunct editing site and ensures translational accuracy.     

A unique insert of leucyl-tRNA synthetase is required for aminoacylation and not amino acid editing

Leucyl-tRNA synthetase (LeuRS) is a class I enzyme, which houses its aminoacylation active site in a canonical core that is defined by a Rossmann nucleotide binding fold. In addition, many LeuRSs bear a unique polypeptide insert comprised of about 50 amino acids located just upstream of the conserved KMSKS sequence. The role of this leucine-specific domain (LS-domain) remains undefined. We hypothesized that this domain may be important for substrate recognition in aminoacylation and/or amino acid editing. We carried out a series of deletion mutations and chimeric swaps within the leucine-specific domain of Escherichia coli. Our results support that the leucine-specific domain is critical for aminoacylation but not required for editing activity. Kinetic analysis determined that deletion of the LS-domain primarily impacts kcat. Because of its proximity to the aminoacylation active site, we propose that this domain interacts with the tRNA during amino acid activation and/or tRNA aminoacylation. Although the leucine-specific domain does not appear to be important to the editing complex, it remains possible that it aids the dynamic translocation process that moves tRNA from the aminoacylation to the editing complex.     

Conformational movements and cooperativity upon amino acid,ATP and tRNA binding in threonyl-tRNA synthetase

The crystal structures of threonyl-tRNA synthetase (ThrRS) from Staphylococcus aureus, with ATP and an analogue of threonyl adenylate, are described. Together with the previously determined structures of Escherichia coli ThrRS with different substrates, they allow a comprehensive analysis of the effect of binding of all the substrates: threonine, ATP and tRNA. The tRNA, by inserting its acceptor arm between the N-terminal domain and the catalytic domain, causes a large rotation of the former. Within the catalytic domain, four regions surrounding the active site display significant conformational changes upon binding of the different substrates. The binding of threonine induces the movement of as much as 50 consecutive amino acid residues. The binding of ATP triggers a displacement, as large as 8A at some C(alpha) positions, of a strand-loop-strand region of the core beta-sheet. Two other regions move in a cooperative way upon binding of threonine or ATP: the motif 2 loop, which plays an essential role in the first step of the aminoacylation reaction, and the ordering loop, which closes on the active site cavity when the substrates are in place. The tRNA interacts with all four mobile regions, several residues initially bound to threonine or ATP switching to a position in which they can contact the tRNA. Three such conformational switches could be identified, each of them in a different mobile region. The structural analysis suggests that, while the small substrates can bind in any order, they must be in place before productive tRNA binding can occur.     

The crystal structure of the ternary complex of T.thermophilus seryl-tRNA synthetase with tRNA(Ser) and a seryl-adenylate analogue reveals a conformational switch in the active site.

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.     

A glycine hinge for tRNA-dependent translocation of editing substrates to prevent errors by leucyl-tRNA synthetase

Aminoacyl-tRNA synthetases often rely on a proofreading mechanism to clear mischarging errors before they can be incorporated into newly synthesized proteins. Leucyl-tRNA synthetase (LeuRS) houses a hydrolytic editing pocket in a domain that is distinct from its aminoacylation domain. Mischarged amino acids are transiently translocated ∼30 Å between active sites for editing by an unknown tRNA-dependent mechanism. A glycine within a flexible β-strand that links the aminoacylation and editing domains of LeuRS was determined to be important to tRNA translocation. The translocation-defective mutation also demonstrated that the editing site screens both correctly and incorrectly charged tRNAs prior to product release.     

Allosteric interaction of nucleotides and tRNA(ala) with E. coli alanyl-tRNA synthetase

Alanyl-tRNA synthetase, a dimeric class 2 aminoacyl-tRNA synthetase, activates glycine and serine at significant rates. An editing activity hydrolyzes Gly-tRNA(ala) and Ser-tRNA(ala) to ensure fidelity of aminoacylation. Analytical ultracentrifugation demonstrates that the enzyme is predominately a dimer in solution. ATP binding to full length enzyme (ARS875) and to an N-terminal construct (ARS461) is endothermic (ΔH = 3-4 kcal mol(-1)) with stoichiometries of 1:1 for ARS461 and 2:1 for full-length dimer. Binding of aminoacyl-adenylate analogues, 5'-O-[N-(L-alanyl)sulfamoyl]adenosine (ASAd) and 5'-O-[N-(L-glycinyl)sulfamoyl]adenosine (GSAd), are exothermic; ASAd exhibits a large negative heat capacity change (ΔC(p) = 0.48 kcal mol(-1) K(-1)). Modification of alanyl-tRNA synthetase with periodate-oxidized tRNA(ala) (otRNA(ala)) generates multiple, covalent, enzyme-tRNA(ala) products. The distribution of these products is altered by ATP, ATP and alanine, and aminoacyl-adenylate analogues (ASAd and GSAd). Alanyl-tRNA synthetase was modified with otRNA(ala), and tRNA-peptides from tryptic digests were purified by ion exchange chromatography. Six peptides linked through a cyclic dehydromoropholino structure at the 3'-end of tRNA(ala) were sequenced by mass spectrometry. One site lies in the N-terminal adenylate synthesis domain (residue 74), two lie in the opening to the editing site (residues 526 and 585), and three (residues 637, 639, and 648) lie on the back side of the editing domain. At least one additional modification site was inferred from analysis of modification of ARS461. The location of the sites modified by otRNA(ala) suggests that there are multiple modes of interaction of tRNA(ala) with the enzyme, whose distribution is influenced by occupation of the ATP binding site.     

The crystal structure of leucyl-tRNA synthetase complexed with tRNALeu in the post-transfer-editing conformation

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.