Substrate-mediated Fidelity Mechanism Ensures Accurate Decoding of Proline Codons

Aminoacyl-tRNA synthetases attach specific amino acids to cognate tRNAs. Prolyl-tRNA synthetases are known to mischarge tRNA^Pro with the smaller amino acid alanine and with cysteine, which is the same size as proline. Quality control in proline codon translation is partly ensured by an editing domain (INS) present in most bacterial prolyl-tRNA synthetases that hydrolyzes smaller Ala-tRNA^Pro and excludes Pro-tRNA^Pro. In contrast, Cys-tRNA^Pro is cleared by a freestanding INS domain homolog, YbaK. Here, we have investigated the molecular mechanism of catalysis and substrate recognition by Hemophilus influenzae YbaK using site-directed mutagenesis, enzymatic assays of isosteric substrates and functional group analogs, and computational modeling. These studies together with mass spectrometric characterization of the YbaK-catalyzed reaction products support a novel substrate-assisted mechanism of Cys-tRNA^Pro deacylation that prevents nonspecific Pro-tRNA^Pro hydrolysis. Collectively, we propose that the INS and YbaK domains co-evolved distinct mechanisms involving steric exclusion and thiol-specific chemistry, respectively, to ensure accurate decoding of proline codons.     

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.     

Kinetic Origin of Substrate Specificity in Post-Transfer Editing by Leucyl-tRNA Synthetase

The intrinsic editing capacities of aminoacyl-tRNA synthetases ensure a high-fidelity translation of the amino acids that possess effective non-cognate aminoacylation surrogates. The dominant error-correction pathway comprises deacylation of misaminoacylated tRNA within the aminoacyl-tRNA synthetase editing site. To assess the origin of specificity of Escherichia coli leucyl-tRNA synthetase (LeuRS) against the cognate aminoacylation product in editing, we followed binding and catalysis independently using cognate leucyl- and non-cognate norvalyl-tRNA^Leu and their non-hydrolyzable analogues. We found that the amino acid part (leucine versus norvaline) of (mis)aminoacyl-tRNAs can contribute approximately 10-fold to ground-state discrimination at the editing site. In sharp contrast, the rate of deacylation of leucyl- and norvalyl-tRNA^Leu differed by about 10⁴-fold. We further established the critical role for the A76 3′-OH group of the tRNA^Leu in post-transfer editing, which supports the substrate-assisted deacylation mechanism. Interestingly, the abrogation of the LeuRS specificity determinant threonine 252 did not improve the affinity of the editing site for the cognate leucine as expected, but instead substantially enhanced the rate of leucyl-tRNA^Leu hydrolysis. In line with that, molecular dynamics simulations revealed that the wild-type enzyme, but not the T252A mutant, enforced leucine to adopt the side-chain conformation that promotes the steric exclusion of a putative catalytic water. Our data demonstrated that the LeuRS editing site exhibits amino acid specificity of kinetic origin, arguing against the anticipated prominent role of steric exclusion in the rejection of leucine. This feature distinguishes editing from the synthetic site, which relies on ground-state discrimination in amino acid selection.     

Distinct tRNA recognition strategies used by a homologous family of editing domains prevent mistranslation

Errors in protein synthesis due to mispairing of amino acids with tRNAs jeopardize cell viability. Several checkpoints to prevent formation of Ala- and Cys-tRNA^Pro have been described, including the Ala-specific editing domain (INS) of most bacterial prolyl-tRNA synthetases (ProRSs) and an autonomous single-domain INS homolog, YbaK, which clears Cys-tRNA^Pro in trans. In many species where ProRS lacks an INS domain, ProXp-ala, another single-domain INS-like protein, is responsible for editing Ala-tRNA^Pro. Although the amino acid specificity of these editing domains has been established, the role of tRNA sequence elements in substrate selection has not been investigated in detail. Critical recognition elements for aminoacylation by bacterial ProRS include acceptor stem elements G72/A73 and anticodon bases G35/G36. Here, we show that ProXp-ala and INS require these same acceptor stem and anticodon elements, respectively, whereas YbaK lacks inherent tRNA specificity. Thus, these three related domains use divergent approaches to recognize tRNAs and prevent mistranslation. Whereas some editing domains have borrowed aspects of tRNA recognition from the parent aminoacyl-tRNA synthetase, relaxed tRNA specificity leading to semi-promiscuous editing may offer advantages to cells.     

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.     

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.     

Human trans-editing enzyme displays tRNA acceptor-stem specificity and relaxed amino acid selectivity

Accurate translation of genetic information into proteins is vital for cell sustainability. ProXp-ala prevents proteome-wide Pro-to-Ala mutations by hydrolyzing misacylated Ala-tRNA^Pro, which is synthesized by prolyl-tRNA synthetase. Bacterial ProXp-ala was previously shown to combine a size-based exclusion mechanism with conformational and chemical selection for the recognition of the alanyl moiety, whereas tRNA^Pro is selected via recognition of tRNA acceptor-stem elements G72 and A73. The identity of these critical bases changed during evolution with eukaryotic cytosolic tRNA^Pro possessing a cytosine at the corresponding positions. The mechanism by which eukaryotic ProXp-ala adapted to these changes remains unknown. In this work, recognition of the aminoacyl moiety and tRNA acceptor stem by human (Homo sapiens, or Hs) ProXp-ala was examined. Enzymatic assays revealed that Hs ProXp-ala requires C72 and C73 in the context of Hs cytosolic tRNA^Pro for efficient deacylation of mischarged Ala-tRNA^Pro. The strong dependence on these bases prevents cross-species deacylation of bacterial Ala-tRNA^Pro or of Hs mitochondrial Ala-tRNA^Pro by the human enzyme. Similar to the bacterial enzyme, Hs ProXp-ala showed strong tRNA acceptor-stem recognition but differed in its amino acid specificity profile relative to bacterial ProXp-ala. Changes at conserved residues in both the Hs and bacterial ProXp-ala substrate-binding pockets modulated this specificity. These results illustrate how the mechanism of substrate selection diverged during the evolution of the ProXp-ala family, providing the first example of a trans-editing domain whose specificity evolved to adapt to changes in its tRNA substrate.     

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.     

Species-specific differences in amino acid editing by class II prolyl-tRNA synthetase.

Aminoacyl-tRNA synthetases are a family of enzymes responsible for ensuring the accuracy of the genetic code by specifically attaching a particular amino acid to their cognate tRNA substrates. Through primary sequence alignments, prolyl-tRNA synthetases (ProRSs) have been divided into two phylogenetically divergent groups. We have been interested in understanding whether the unusual evolutionary pattern of ProRSs corresponds to functional differences as well. Previously, we showed that some features of tRNA recognition and aminoacylation are indeed group-specific. Here, we examine the species-specific differences in another enzymatic activity, namely amino acid editing. Proofreading or editing provides a mechanism by which incorrectly activated amino acids are hydrolyzed and thus prevented from misincorporation into proteins. "Prokaryotic-like" Escherichia coli ProRS has recently been shown to be capable of misactivating alanine and possesses both pretransfer and post-transfer hydrolytic editing activity against this noncognate amino acid. We now find that two ProRSs belonging to the "eukaryotic-like" group exhibit differences in their hydrolytic editing activity. Whereas ProRS from Methanococcus jannaschii is similar to E. coli in its ability to hydrolyze misactivated alanine via both pretransfer and post-transfer editing pathways, human ProRS lacks these activities. These results have implications for the selection or design of antibiotics that specifically target the editing active site of the prokaryotic-like group of ProRSs.     

Structural modelling of the complex of leucyl-tRNA synthetase and mis-aminoacylated tRNA

To assure fidelity of translation, class Ia aminoacyl-tRNA synthetases (aaRSs) edit mis-aminoacylated tRNAs. Mis-attached amino acids and structural water molecules are not included simultaneously in the current crystal structures of the aaRS•tRNA complexes, where the 3′-ends (adenine 76; A76) are bound to the editing sites. A structural model of the completely solvated leucyl-tRNA synthetase complexed with valyl-tRNA^Leu was constructed by exploiting molecular dynamics simulations modified for the present modelling. The results showed that the ribose conformation of A76 is distinct from those observed in the above-mentioned crystal structures, which could be derived from structural constraints in a sandwiched manner induced by the mis-attached valine and tRNA^Leu.     

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.     

Kinetic partitioning between synthetic and editing pathways in class I aminoacyl-tRNA synthetases occurs at both pre-transfer and post-transfer hydrolytic steps

Comprehensive steady-state and transient kinetic studies of the synthetic and editing activities of Escherichia coli leucyl-tRNA synthetase (LeuRS) demonstrate that the enzyme depends almost entirely on post-transfer editing to endow the cell with specificity against incorporation of norvaline into protein. Among the three class I tRNA synthetases possessing a dedicated post-transfer editing domain (connective peptide 1; CP1 domain), LeuRS resembles valyl-tRNA synthetase in its reliance on post-transfer editing, whereas isoleucyl-tRNA synthetase differs in retaining a distinct tRNA-dependent synthetic site pre-transfer editing activity to clear noncognate amino acids before misacylation. Further characterization of the post-transfer editing activity in LeuRS by single-turnover kinetics demonstrates that the rate-limiting step is dissociation of deacylated tRNA and/or amino acid product and highlights the critical role of a conserved aspartate residue in mediating the first-order hydrolytic steps on the enzyme. Parallel analyses of adenylate and aminoacyl-tRNA formation reactions by wild-type and mutant LeuRS demonstrate that the efficiency of post-transfer editing is controlled by kinetic partitioning between hydrolysis and dissociation of misacylated tRNA and shows that trans editing after rebinding is a competent kinetic pathway. Together with prior analyses of isoleucyl-tRNA synthetase and valyl-tRNA synthetase, these experiments provide the basis for a comprehensive model of editing by class I tRNA synthetases, in which kinetic partitioning plays an essential role at both pre-transfer and post-transfer steps.     

tRNA-dependent Pre-transfer Editing by Prokaryotic Leucyl-tRNA Synthetase

To prevent genetic code ambiguity due to misincorporation of amino acids into proteins, aminoacyl-tRNA synthetases have evolved editing activities to eliminate intermediate or final non-cognate products. In this work we studied the different editing pathways of class Ia leucyl-tRNA synthetase (LeuRS). Different mutations and experimental conditions were used to decipher the editing mechanism, including the recently developed compound AN2690 that targets the post-transfer editing site of LeuRS. The study emphasizes the crucial importance of tRNA for the pre- and post-transfer editing catalysis. Both reactions have comparable efficiencies in prokaryotic Aquifex aeolicus and Escherichia coli LeuRSs, although the E. coli enzyme favors post-transfer editing, whereas the A. aeolicus enzyme favors pre-transfer editing. Our results also indicate that the entry of the CCA-acceptor end of tRNA in the editing domain is strictly required for tRNA-dependent pre-transfer editing. Surprisingly, this editing reaction was resistant to AN2690, which inactivates the enzyme by forming a covalent adduct with tRNA^Leu in the post-transfer editing site. Taken together, these data suggest that the binding of tRNA in the post-transfer editing conformation confers to the enzyme the capacity for pre-transfer editing catalysis, regardless of its capacity to catalyze post-transfer editing.     

In vitro assays for the determination of aminoacyl-tRNA synthetase editing activity

Aminoacyl-tRNA synthetases are essential enzymes that help to ensure the fidelity of protein translation by accurately aminoacylating (or "charging") specific tRNA substrates with cognate amino acids. Many synthetases have an additional catalytic activity to confer amino acid editing or proofreading. This activity relieves ambiguities during translation of the genetic code that result from one synthetase activating multiple amino acid substrates. In this review, we describe methods that have been developed for assaying both pre- and post-transfer editing activities. Pre-transfer editing is defined as hydrolysis of a misactivated aminoacyl-adenylate prior to transfer to the tRNA. This reaction has been reported to occur either in the aminoacylation active site or in a separate editing domain. Post-transfer editing refers to the hydrolysis reaction that cleaves the aminoacyl-ester linkage formed between the carbonyl carbon of the amino acid and the 2' or 3' hydroxyl group of the ribose on the terminal adenosine. Post-transfer editing takes place in a hydrolytic active site that is distinct from the site of amino acid activation. Here, we focus on methods for determination of steady-state reaction rates using editing assays developed for both classes of synthetases.     

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.     

Alternative pathways for editing non-cognate amino acids by aminoacyl-tRNA synthetases.

Evidence is presented that the editing mechanisms of aminoacyl-tRNA synthetase operate by two alternative pathways: pre-transfer, by hydrolysis of the non-cognate aminoacyl adenylate; post-transfer, by hydrolysis of the mischarged tRNA. The methionyl-tRNA synthetases from Escherichia coli and Bacillus stearothermophilus and isoleucyl-tRNA synthetase from E. coli, for example, are shown to reject misactivated homocysteine rapidly by the pre-transfer route. A novel feature of this reaction is that homocysteine thiolactone is formed by the facile cyclisation of the homocysteinyl adenylate. Valyl-tRNA synthetases, on the other hand, reject the more readily activated non-cognate amino acids by primarily the post-transfer route. The features governing the choice of pathway are discussed.     

The Yin and Yang of tRNA: proper binding of acceptor end determines the catalytic balance of editing and aminoacylation

Faithful translation of the genetic code depends on accurate coupling of amino acids with cognate transfer RNAs (tRNAs) catalyzed by aminoacyl-tRNA synthetases. The fidelity of leucyl-tRNA synthetase (LeuRS) depends mainly on proofreading at the pre- and post-transfer levels. During the catalytic cycle, the tRNA CCA-tail shuttles between the synthetic and editing domains to accomplish the aminoacylation and editing reactions. Previously, we showed that the Y330D mutation of Escherichia coli LeuRS, which blocks the entry of the tRNA CCA-tail into the connective polypeptide 1domain, abolishes both tRNA-dependent pre- and post-transfer editing. In this study, we identified the counterpart substitutions, which constrain the tRNA acceptor stem binding within the synthetic active site. These mutations negatively impact the tRNA charging activity while retaining the capacity to activate the amino acid. Interestingly, the mutated LeuRSs exhibit increased global editing activity in the presence of a non-cognate amino acid. We used a reaction mimicking post-transfer editing to show that these mutations decrease post-transfer editing owing to reduced tRNA aminoacylation activity. This implied that the increased editing activity originates from tRNA-dependent pre-transfer editing. These results, together with our previous work, provide a comprehensive assessment of how intra-molecular translocation of the tRNA CCA-tail balances the aminoacylation and editing activities of LeuRS.     

Aminoacyl Transfer Rate Dictates Choice of Editing Pathway in Threonyl-tRNA Synthetase

Aminoacyl-tRNA synthetases hydrolyze aminoacyl adenylates and aminoacyl-tRNAs formed from near-cognate amino acids, thereby increasing translational fidelity. The contributions of pre- and post-transfer editing pathways to the fidelity of Escherichia coli threonyl-tRNA synthetase (ThrRS) were investigated by rapid kinetics. In the pre-steady state, asymmetric activation of cognate threonine and noncognate serine was observed in the active sites of dimeric ThrRS, with similar rates of activation. In the absence of tRNA, seryl-adenylate was hydrolyzed 29-fold faster by the ThrRS catalytic domain than threonyl-adenylate. The rate of seryl transfer to cognate tRNA was only 2-fold slower than threonine. Experiments comparing the rate of ATP consumption to the rate of aminoacyl-tRNA^AA formation demonstrated that pre-transfer hydrolysis contributes to proofreading only when the rate of transfer is slowed significantly. Thus, the relative contributions of pre- and post-transfer editing in ThrRS are subject to modulation by the rate of aminoacyl transfer.     

Modular pathways for editing non-cognate amino acids by human cytoplasmic leucyl-tRNA synthetase

To prevent potential errors in protein synthesis, some aminoacyl-transfer RNA (tRNA) synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated tRNAs (post-transfer editing). Class Ia leucyl-tRNA synthetase (LeuRS) may misactivate various natural and non-protein amino acids and then mischarge tRNA^Leu. It is known that the fidelity of prokaryotic LeuRS depends on multiple editing pathways to clear the incorrect intermediates and products in the every step of aminoacylation reaction. Here, we obtained human cytoplasmic LeuRS (hcLeuRS) and tRNA^Leu (hctRNA^Leu) with high activity from Escherichia coli overproducing strains to study the synthetic and editing properties of the enzyme. We revealed that hcLeuRS could adjust its editing strategy against different non-cognate amino acids. HcLeuRS edits norvaline predominantly by post-transfer editing; however, it uses mainly pre-transfer editing to edit α-amino butyrate, although both amino acids can be charged to tRNA^Leu. Post-transfer editing as a final checkpoint of the reaction was very important to prevent mis-incorporation in vitro. These results provide insight into the modular editing pathways created to prevent genetic code ambiguity by evolution.     

An isolated class II aminoacyl-tRNA synthetase insertion domain is functional in amino acid editing

Aminoacyl-tRNA synthetases are responsible for activating specific amino acids and transferring them onto cognate tRNA molecules. Due to the similarity in many amino acid side chains, certain synthetases misactivate non-cognate amino acids to an extent that would be detrimental to protein synthesis if left uncorrected. To ensure accurate translation of the genetic code, some synthetases therefore utilize editing mechanisms to hydrolyze non-cognate products. Previously class II Escherichia coli proline-tRNA synthetase (ProRS) was shown to exhibit pre- and post-transfer editing activity, hydrolyzing a misactivated alanine-adenylate (Ala-AMP) and a mischarged Ala-tRNAPro variant, respectively. Residues critical for the editing activity (Asp-350 and Lys-279) are found in a novel insertion domain (INS) positioned between motifs 2 and 3 of the class defining aminoacylation active site. In this work, we present further evidence that INS is responsible for editing in ProRS. We deleted the INS from wild-type E. coli ProRS to yield DeltaINS-ProRS. While DeltaINS-ProRS was still capable of misactivating alanine, the truncated construct was defective in hydrolyzing non-cognate Ala-AMP. When the INS domain was cloned and expressed as an independent protein, it was capable of deacylating a mischarged Ala-microhelixPro variant. Similar to full-length ProRS, post-transfer editing was abolished in a K279A mutant INS. We also show that YbaK, a protein of unknown function from Haemophilus influenzae with high sequence homology to the prokaryotic INS domain, was capable of deacylating Ala-tRNAPro and Ala-microhelixPro variants but not cognate Pro-tRNAPro. Thus, we demonstrate for the first time that an independently folded class II synthetase editing domain and a previously identified homolog can catalyze a hydrolytic editing reaction.