The tRNA A76 Hydroxyl Groups Control Partitioning of the tRNA-dependent Pre- and Post-transfer Editing Pathways in Class I tRNA Synthetase

Aminoacyl-tRNA synthetases catalyze ATP-dependent covalent coupling of cognate amino acids and tRNAs for ribosomal protein synthesis. Escherichia coli isoleucyl-tRNA synthetase (IleRS) exploits both the tRNA-dependent pre- and post-transfer editing pathways to minimize errors in translation. However, the molecular mechanisms by which tRNA^Ile organizes the synthetic site to enhance pre-transfer editing, an idiosyncratic feature of IleRS, remains elusive. Here we show that tRNA^Ile affects both the synthetic and editing reactions localized within the IleRS synthetic site. In a complex with cognate tRNA, IleRS exhibits a 10-fold faster aminoacyl-AMP hydrolysis and a 10-fold drop in amino acid affinity relative to the free enzyme. Remarkably, the specificity against non-cognate valine was not improved by the presence of tRNA in either of these processes. Instead, amino acid specificity is determined by the protein component per se, whereas the tRNA promotes catalytic performance of the synthetic site, bringing about less error-prone and kinetically optimized isoleucyl-tRNA^Ile synthesis under cellular conditions. Finally, the extent to which tRNA^Ile modulates activation and pre-transfer editing is independent of the intactness of its 3′-end. This finding decouples aminoacylation and pre-transfer editing within the IleRS synthetic site and further demonstrates that the A76 hydroxyl groups participate in post-transfer editing only. The data are consistent with a model whereby the 3′-end of the tRNA remains free to sample different positions within the IleRS·tRNA complex, whereas the fine-tuning of the synthetic site is attained via conformational rearrangement of the enzyme through the interactions with the remaining parts of the tRNA body.     

A present-day aminoacyl-tRNA synthetase with ancestral editing properties

Leucyl-, isoleucyl-, and valyl-tRNA synthetases form a subgroup of related aminoacyl-tRNA synthetases that attach similar amino acids to their cognate tRNAs. To prevent amino acid misincorporation during translation, these enzymes also hydrolyze mischarged tRNAs through a post-transfer editing mechanism. Here we show that LeuRS from the deep-branching bacterium Aquifex aeolicus edits the complete set of aminoacylated tRNAs generated by the three enzymes: Ile-tRNA(Ile), Val-tRNA(Ile), Val-tRNA(Val), Thr-tRNA(Val), and Ile-tRNA(Leu). This unusual enlarged editing property was studied in a model of a primitive editing system containing a composite minihelix carrying the triple leucine, isoleucine, and valine identity mimicking the primitive tRNA precursor. We found that the freestanding LeuRS editing domain can edit this precursor in contrast to IleRS and ValRS editing domains. These results suggest that A. aeolicus LeuRS carries editing properties that seem more primitive than those of IleRS and ValRS. They suggest that the A. aeolicus editing domain has preserved the ambiguous editing property from the ancestral common editing domain or, alternatively, that this plasticity results from a specific metabolic adaptation.     

Negative catalysis by the editing domain of class I aminoacyl-tRNA synthetases

Aminoacyl-tRNA synthetases (AARS) translate the genetic code by loading tRNAs with the cognate amino acids. The errors in amino acid recognition are cleared at the AARS editing domain through hydrolysis of misaminoacyl-tRNAs. This ensures faithful protein synthesis and cellular fitness. Using Escherichia coli isoleucyl-tRNA synthetase (IleRS) as a model enzyme, we demonstrated that the class I editing domain clears the non-cognate amino acids well-discriminated at the synthetic site with the same rates as the weakly-discriminated fidelity threats. This unveiled low selectivity suggests that evolutionary pressure to optimize the rates against the amino acids that jeopardize translational fidelity did not shape the editing site. Instead, we propose that editing was shaped to safeguard cognate aminoacyl-tRNAs against hydrolysis. Misediting is prevented by the residues that promote negative catalysis through destabilisation of the transition state comprising cognate amino acid. Such powerful design allows broad substrate acceptance of the editing domain along with its exquisite specificity in the cognate aminoacyl-tRNA rejection. Editing proceeds by direct substrate delivery to the editing domain (in cis pathway). However, we found that class I IleRS also releases misaminoacyl-tRNA^Ile and edits it in trans. This minor editing pathway was up to now recognized only for class II AARSs.     

Plasticity of recognition of the 3'-end of mischarged tRNA by class I aminoacyl-tRNA synthetases

Certain aminoacyl-tRNA synthetases prevent potential errors in protein synthesis through deacylation of mischarged tRNAs. For example, the close homologs isoleucyl-tRNA synthetase (IleRS) and valyl-tRNA synthetase (ValRS) deacylate Val-tRNA(Ile) and Thr-tRNA(Val), respectively. Here we examined the chemical requirements at the 3'-end of the tRNA for these hydrolysis reactions. Single atom substitutions at the 2'- and 3'-hydroxyls of a variety of mischarged RNAs revealed that, while acylation is at the 2'-OH for both enzymes, IleRS catalyzes deacylation specifically from the 3'-OH and not from the 2'-OH. In contrast, ValRS can deacylate non-cognate amino acids from the 2'-OH. Moreover, for IleRS the specificity for a 3'-O location of the scissile ester bond could be forced to the 2'-position by introduction of a 3'-O-methyl moiety. Cumulatively, these and other results suggest that the editing sites of these class I aminoacyl-tRNA synthetases have a degree of inherent plasticity for substrate recognition. The ability to adapt to subtle differences in mischarged RNAs may be important for the high accuracy of aminoacylation.     

Partitioning of tRNA-dependent Editing between Pre- and Post-transfer Pathways in Class I Aminoacyl-tRNA Synthetases

Hydrolytic editing activities are present in aminoacyl-tRNA synthetases possessing reduced amino acid discrimination in the synthetic reactions. Post-transfer hydrolysis of misacylated tRNA in class I editing enzymes occurs in a spatially separate domain inserted into the catalytic Rossmann fold, but the location and mechanisms of pre-transfer hydrolysis of misactivated amino acids have been uncertain. Here, we use novel kinetic approaches to distinguish among three models for pre-transfer editing by Escherichia coli isoleucyl-tRNA synthetase (IleRS). We demonstrate that tRNA-dependent hydrolysis of noncognate valyl-adenylate by IleRS is largely insensitive to mutations in the editing domain of the enzyme and that noncatalytic hydrolysis after release is too slow to account for the observed rate of clearing. Measurements of the microscopic rate constants for amino acid transfer to tRNA in IleRS and the related valyl-tRNA synthetase (ValRS) further suggest that pre-transfer editing in IleRS is an enzyme-catalyzed activity residing in the synthetic active site. In this model, the balance between pre-transfer and post-transfer editing pathways is controlled by kinetic partitioning of the noncognate aminoacyl-adenylate. Rate constants for hydrolysis and transfer of a noncognate intermediate are roughly equal in IleRS, whereas in ValRS transfer to tRNA is 200-fold faster than hydrolysis. In consequence, editing by ValRS occurs nearly exclusively by post-transfer hydrolysis in the editing domain, whereas in IleRS both pre- and post-transfer editing are important. In both enzymes, the rates of amino acid transfer to tRNA are similar for cognate and noncognate aminoacyl-adenylates, providing a significant contrast with editing DNA polymerases.     

Modulation of substrate specificity within the amino acid editing site of leucyl-tRNA synthetase

The aminoacyl-tRNA synthetases covalently link transfer RNAs to their cognate amino acids. Some of the tRNA synthetases have evolved editing mechanisms to ensure fidelity in this first step of protein synthesis. The amino acid editing site for leucyl- (LeuRS) and isoleucyl- (IleRS) tRNA synthetases reside within homologous CP1 domains. In each case, a threonine-rich peptide and a second conserved GTG region that are separated by about 100 amino acids comprise parts of the hydrolytic editing site. While a number of sites are conserved between these two enzymes and likely confer a commonality to the mechanisms, some positions are idiosyncratic to LeuRS or IleRS. Herein, we provide evidence that a conserved arginine and threonine at respective sites in LeuRS and IleRS diverged to confer amino acid substrate recognition. This site complements other sites in the amino acid binding pocket of the editing active site of Escherichia coli LeuRS, including Thr252 and Val338, which collectively fine-tune amino acid specificity to confer fidelity.     

Structural basis for substrate recognition by the editing domain of isoleucyl-tRNA synthetase

In isoleucyl-tRNA synthetase (IleRS), the "editing" domain contributes to accurate aminoacylation by hydrolyzing the mis-synthesized intermediate, valyl-adenylate, in the "pre-transfer" editing mode and the incorrect final product, valyl-tRNA(Ile), in the "post-transfer" editing mode. In the present study, we determined the crystal structures of the Thermus thermophilus IleRS editing domain complexed with the substrate analogues in the pre and post-transfer modes, both at 1.7 A resolution. The active site accommodates the two analogues differently, with the valine side-chain rotated by about 120 degrees and the adenosine moiety oriented upside down. The substrate-binding pocket adjusts to the adenosine-monophosphate and adenosine moieties in the pre and post-transfer modes, respectively, by flipping the Trp227 side-chain by about 180 degrees . The substrate recognition mechanisms of IleRS are characterized by the active-site rearrangement between the two editing modes, and therefore differ from those of the homologous valyl and leucyl-tRNA synthetases from T.thermophilus, in which the post-transfer mode is predominant. Both modes of editing activities were reduced by replacements of Trp227 with Ala, Val, Leu, and His, but not by those with Phe and Tyr, indicating that the aromatic ring of Trp227 is important for the substrate recognition. In both editing modes, Thr233 and His319 recognize the substrate valine side-chain, regardless of the valine side-chain rotation, and reject the isoleucine side-chain. The T233A and H319A mutants have detectable editing activities against the cognate isoleucine.     

Kinetic Proofreading at Single Molecular Level: Aminoacylation of tRNAIle and the Role of Water as an Editor

Proofreading/editing in protein synthesis is essential for accurate translation of information from the genetic code. In this article we present a theoretical investigation of efficiency of a kinetic proofreading mechanism that employs hydrolysis of the wrong substrate as the discriminatory step in enzyme catalytic reactions. We consider aminoacylation of tRNA^Ile which is a crucial step in protein synthesis and for which experimental results are now available. We present an augmented kinetic scheme and then employ methods of stochastic simulation algorithm to obtain time dependent concentrations of different substances involved in the reaction and their rates of formation. We obtain the rates of product formation and ATP hydrolysis for both correct and wrong substrates (isoleucine and valine in our case, respectively), in single molecular enzyme as well as ensemble enzyme kinetics. The present theoretical scheme correctly reproduces (i) the amplitude of the discrimination factor in the overall rates between isoleucine and valine which is obtained as (1.8×10²).(4.33×10²) = 7.8×10⁴, (ii) the rates of ATP hydrolysis for both Ile and Val at different substrate concentrations in the aminoacylation of tRNA^Ile. The present study shows a non-michaelis type dependence of rate of reaction on tRNA^Ile concentration in case of valine. The overall editing in steady state is found to be independent of amino acid concentration. Interestingly, the computed ATP hydrolysis rate for valine at high substrate concentration is same as the rate of formation of Ile-tRNA^Ile whereas at intermediate substrate concentration the ATP hydrolysis rate is relatively low. We find that the presence of additional editing domain in class I editing enzyme makes the kinetic proofreading more efficient through enhanced hydrolysis of wrong product at the editing CP1 domain.     

Crystal structures of the CP1 domain from Thermus thermophilus isoleucyl-tRNA synthetase and its complex with L-valine

Isoleucyl-tRNA synthetase (IleRS) links tRNA(Ile) with not only its cognate isoleucine but also the nearly cognate valine. The CP1 domain of IleRS deacylates, or edits, the mischarged Val-tRNA(Ile). We determined the crystal structures of the Thermus thermophilus IleRS CP1 domain alone, and in its complex with valine at 1.8- and 2.0-A resolutions, respectively. In the complex structure, the Asp(328) residue, which was shown to be critical for the editing reaction against Val-tRNA(Ile) by a previous mutational analysis, recognizes the valine NH(3)(+) group. The valine side chain binding pocket is only large enough to accommodate valine, and the placement of an isoleucine model in this location revealed that the additional methylene group of isoleucine would clash with His(319). The H319A mutant of Escherichia coli IleRS reportedly deacylates the cognate Ile-tRNA(Ile) in addition to Val-tRNA(Ile), indicating that the valine-binding mode found in this study represents that in the Val-tRNA(Ile) editing reaction. Analyses of the Val-tRNA(Ile) editing activities of T. thermophilus IleRS mutants revealed the importance of Thr(228), Thr(229), Thr(230), and Asp(328), which are coordinated with water molecules in the present structure. The structural model for the Val-adenosine moiety of Val-tRNA(Ile) bound in the IleRS editing site revealed some interesting differences in the substrate binding and recognizing mechanisms between IleRS and T. thermophilus leucyl-tRNA synthetase. For example, the carbonyl oxygens of the amino acids are located opposite to each other, relative to the adenosine ribose ring, and are differently recognized.     

Crystal structure of leucyl-tRNA synthetase from the archaeon Pyrococcus horikoshii reveals a novel editing domain orientation

The editing domains of the closely homologous leucyl, isoleucyl, and valyl-tRNA synthetases (LeuRS, IleRS, and ValRS, respectively) contribute to accurate aminoacylation, by hydrolyzing misformed non-cognate aminoacyl-tRNAs. The editing domain is inserted at the same point of the sequence in IleRS, ValRS, and the archaeal/eukaryal LeuRS, but at a distinct point in the bacterial LeuRS. Here, we showed that LeuRS from the archaeon Pyrococcus horikoshii has editing activity against the nearly cognate isoleucine. The conserved Asp332 in the editing domain is crucial for this activity. A deletion mutant lacking the C-terminal region has only negligible aminoacylation activity, but retains the full activity of adenylate synthesis and editing. We determined the crystal structure of this editing-active, truncated form of P.horikoshii LeuRS at 2.1 A resolution. The structure revealed that it has a novel editing domain orientation. The editing domain of P.horikoshii LeuRS is rotated by approximately 180 degrees (rotational state II), with the two-beta-stranded linker untwisted by a half-turn, as compared to those in IleRS and ValRS (rotational state I). This editing domain rotational state in the archaeal LeuRS is similar to that in the bacterial LeuRS. However, because of the insertion point difference, the orientation of the editing domain relative to the enzyme core in the archaeal LeuRS differs completely from that in the bacterial LeuRS. An insertion region specific to the archaeal/eukaryal LeuRS editing domains interacts with the enzyme core and stabilizes the unique orientation. Thus, we established that there are three types of editing domain orientations relative to the enzyme core, depending on the combination of the editing domain insertion point (i or ii) and the rotational state (I or II): [i, I] for IleRS and ValRS, [ii, II] for the bacterial LeuRS, and now [i, II] for the archaeal/eukaryal LeuRS.     

The C-terminal domain of the archaeal leucyl-tRNA synthetase prevents misediting of isoleucyl-tRNA(Ile)

In the archaeal leucyl-tRNA synthetase (LeuRS), the C-terminal domain recognizes the long variable arm of tRNA(Leu) for aminoacylation, and the so-called editing domain deacylates incorrectly formed Ile-tRNA(Leu). We previously reported, for Pyrococcus horikoshii LeuRS, that a deletion mutant lacking the C-terminal domain (LeuRS_delta(811-967)) retains normal editing activity, but has severely reduced aminoacylation activity. In this study, we found that LeuRS_delta(811-967), but not the wild-type LeuRS, exhibited surprisingly robust deacylation activity against Ile-tRNA(Ile), correctly formed by isoleucyl-tRNA synthetase ("misediting"). Structural superposition of tRNA(Ile) onto the LeuRS x tRNA(Leu) complex indicated that Ile911, Lys912, and Glu913 of the LeuRS C-terminal domain clash with U20 of tRNA(Ile), which is bulged out as compared to the corresponding nucleotide of tRNA(Leu). The deletion of amino acid residues 911-913 of LeuRS enhanced the Ile-tRNA(Ile) deacylation activity, without affecting the Ile-tRNA(Leu) deacylation activity. These results demonstrate that the clashing between U20 of tRNA(Ile) and residues 911-913 of the LeuRS C-terminal domain is the structural mechanism that prevents misediting. In contrast, the deletion of the C-terminal domains of the isoleucyl- and valyl-tRNA synthetases impaired both the aminoacylation (Ile-tRNA(Ile) and Val-tRNA(Val) formation, respectively) and editing (Val-tRNA(Ile) and Thr-tRNA(Val) deacylation, respectively) activities, and did not cause misediting (Val-tRNA(Val) and Thr-tRNA(Thr) deacylation, respectively) activity. Thus, the requirement of the C-terminal domain for misediting prevention is unique to LeuRS, which does not recognize the anticodon of the cognate tRNA, unlike the common aminoacyl-tRNA synthetases.     

The yeast model suggests the use of short peptides derived from mt LeuRS for the therapy of diseases due to mutations in several mt tRNAs

We have previously established a yeast model of mitochondrial (mt) diseases. We showed that defective respiratory phenotypes due to point-mutations in mt tRNA^Leu(UUR), tRNA^Ile and tRNA^Val could be relieved by overexpression of both cognate and non-cognate nuclearly encoded mt aminoacyl-tRNA synthetases (aaRS) LeuRS, IleRS and ValRS. More recently, we showed that the isolated carboxy-terminal domain (Cterm) of yeast mt LeuRS, and even short peptides derived from the human Cterm, have the same suppressing abilities as the whole enzymes.In this work, we extend these results by investigating the activity of a number of mt aaRS from either class I or II towards a panel of mt tRNAs. The Cterm of both human and yeast mt LeuRS has the same spectrum of activity as mt aaRS belonging to class I and subclass a, which is the most extensive among the whole enzymes. Yeast Cterm is demonstrated to be endowed with mt targeting activity.Importantly, peptide fragments β30_31 and β32_33, derived from the human Cterm, have even higher efficiency as well as wider spectrum of activity, thus opening new avenues for therapeutic intervention. Bind-shifting experiments show that the β30_31 peptide directly interacts with human mt tRNA^Leu(UUR) and tRNA^Ile, suggesting that the rescuing activity of isolated peptide fragments is mediated by a chaperone-like mechanism.Wide-range suppression appears to be idiosyncratic of LeuRS and its fragments, since it is not shared by Cterminal regions derived from human mt IleRS or ValRS, which are expected to have very different structures and interactions with tRNAs.     

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.     

A N-H nuclear magnetic resonance study on the interaction between isoleucine tRNA and isoleucyl-tRNA synthetase from Escherichia coli

Imino ¹⁵N and ¹H resonances of Escherichia coli tRNA_l^Ile were observed in the absence and presence of E coli isoleucyl-tRNA synthetase. Upon complex formation of tRNA_l^Ile with isoleucyl-tRNA synthetase, some imino ¹⁵N-¹H resonances disappeared, and some others were significantly broadened and/or shifted in the ¹H chemical shift, while the others were observed at the same ¹⁵H-¹H chemical shifts. It was indicated that the binding of tRNA_l^Ile with IleRS affect the following four regions: the anticodon stem, the junction of the acceptor and T stems, the middle of the D stem, and the region where the tertiary base pair connects the T, D, and extra loops. This result is consistent with those of chemical footprinting and site-directed mutagenesis studies. Taken together, these three independent results reveal the recognition mechanism of tRNA_l^Ile by IleRS: IleRS recognizes all the identity determinants distributed throughout the tRNA_l^Ile molecule, which induces changes in the secondary and tertiary structures of tRNA_l^Ile.     

The Val103Ile Polymorphism of Melanocortin‐4 Receptor Regulates Energy Expenditure and Weight Gain

Objective: The melanocortin‐4 receptor (MC4R) regulates energy intake. On the basis of animal studies, it may also regulate energy expenditure. Research Methods and Procedures: The effect of the Val103Ile polymorphism of the MC4R gene on energy metabolism was studied in 229 middle‐aged nondiabetic subjects (Group 1, age 51.2 ± 9.8 years, BMI 26.8 ± 4.5 kg/m²) and on weight gain in 1013 elderly subjects (Group 2, age 69.9 ± 2.9 years, BMI 27.4 ± 4.1 kg/m²) during a 3.5‐year follow‐up study. In Group 1, insulin sensitivity, energy expenditure, and substrate oxidation were measured with the hyperinsulinemic euglycemic clamp combined with indirect calorimetry. Results: In Group 1, the Val103Ile genotype was associated with high rates of energy expenditure (63.42 ± 13.40 in eight subjects with the Val103Ile genotype vs. 59.86 ± 7.33 J/kg per minute in 221 subjects with the Val103Val genotype, p = 0.007), high rates of glucose oxidation (8.90 ± 6.15 vs. 6.07 ± 4.38 μmol/kg per minute, p = 0.020), and low levels of free fatty acids (0.45 ± 0.18 vs. 0.56 ± 0.23 mM, p = 0.029) in the fasting state, and with high rates of glucose oxidation during the clamp (18.88 ± 4.63 vs. 17.60 ± 3.24 μmol/kg per minute, p = 0.031). In Group 2, the 103Ile allele was associated with an increase in weight gain during the follow‐up (0.78 ± 3.98 vs. ?0.82 ± 3.98 kg, p = 0.038). Discussion: The Val103Ile polymorphism of the MC4R gene is associated with energy expenditure in humans. Furthermore, it may associate with glucose oxidation, free fatty acid levels, and weight gain.     

RNA determinants for translational editing. Mischarging a minihelix substrate by a tRNA synthetase

The fidelity of protein synthesis requires efficient discrimination of amino acid substrates by aminoacyl-tRNA synthetases. Accurate discrimination of the structurally similar amino acids, valine and isoleucine, by isoleucyl-tRNA synthetase (IleRS) results, in part, from a hydrolytic editing reaction, which prevents misactivated valine from being stably joined to tRNAIle. The editing reaction is dependent on the presence of tRNAIle, which contains discrete D-loop nucleotides that are necessary to promote editing of misactivated valine. RNA minihelices comprised of just the acceptor-TPsiC helix of tRNAIle are substrates for specific aminoacylation by IleRS. These substrates lack the aforementioned D-loop nucleotides. Because minihelices contain determinants for aminoacylation, we thought that they might also play a role in editing that has not previously been recognized. Here we show that, in contrast to tRNAIle, minihelixIle is unable to trigger the hydrolysis of misactivated valine and, in fact, is mischarged with valine. In addition, mutations in minihelixIle that enhance or suppress charging with isoleucine do the same with valine. Thus, minihelixIle contains signals for charging (by IleRS) that are independent of the amino acid and, by itself, minihelixIle provides no determinants for editing. An RNA hairpin that mimics the D-stem/loop of tRNAIle is also unable to induce the hydrolysis of misactivated valine, both by itself and in combination with minihelixIle. Thus, the native tertiary fold of tRNAIle is required to promote efficient editing. Considering that the minihelix is thought to be the more ancestral part of the tRNA structure, these results are consistent with the idea that, during the development of the genetic code, RNA determinants for editing were added after the establishment of an aminoacylation system.     

Isolated CP1 domain of Escherichia coli leucyl-tRNA synthetase is dependent on flanking hinge motifs for amino acid editing activity

Protein synthesis and its fidelity rely upon the aminoacyl-tRNA synthetases. Leucyl-tRNA synthetase (LeuRS), isoleucyl-tRNA synthetase (IleRS), and valyl-tRNA synthetase (ValRS) have evolved a discrete editing domain called CP1 that hydrolyzes the respective incorrectly misaminoacylated noncognate amino acids. Although active CP1 domain fragments have been isolated for IleRS and ValRS, previous reports suggested that the LeuRS CP1 domain required idiosyncratic adaptations to confer editing activity independent of the full-length enzyme. Herein, characterization of a series of rationally designed Escherichia coli LeuRS fragments showed that the beta-strands, which link the CP1 domain to the aminoacylation core of LeuRS, are required for editing of mischarged tRNALeu. Hydrolytic activity was also enhanced by inclusion of short flexible peptides that have been called "hinges" at the end of both LeuRS beta-strands. We propose that these long beta-strand extensions of the LeuRS CP1 domain interact specifically with the tRNA for post-transfer editing of misaminoacylated amino acids.