A promiscuous aminoacyl-tRNA synthetase that incorporates cysteine, methionine, and alanine homologs into proteins

A mutant Escherichia coli leucyl-tRNA synthetase has been evolved for the selective incorporation of the methionine homolog 1 into proteins in yeast. This single aminoacyl-tRNA synthetase is capable of charging an amber suppressor EctRNA(CUA)(Leu) with at least eight different amino acids including methionine and cysteine homologs, as well as straight chain aliphatic amino acids. In addition we show that incorporation yields for these amino acids can be increased substantially by mutations in the editing CP1 domain of the E. coli leucyl-tRNA synthetase.     

Leucyl-tRNA synthetase from the hyperthermophilic bacterium Aquifex aeolicus recognizes minihelices

Aminoacylation of the minihelix mimicking the amino acid acceptor arm of tRNA has been demonstrated in more than 10 aminoacyl-tRNA synthetase systems. Although Escherichia coli or Homo sapiens cytoplasmic leucyl-tRNA synthetase (LeuRS) is unable to charge the cognate minihelix or microhelix, we show here that minihelix(Leu) is efficiently charged by Aquifex aeolicus synthetase, the only known heterodimeric LeuRS (alpha beta-LeuRS). Aminoacylation of minihelices is strongly dependent on the presence of the A73 identity nucleotide and greatly stimulated by destabilization of the first base pair as reported for the E. coli isoleucyl-tRNA synthetase and methionyl-tRNA synthetase systems. In the E. coli LeuRS system, the anticodon of tRNA(Leu) is not important for recognition by the synthetase. However, the addition of RNA helices that mimic the anticodon domain stimulates minihelix(Leu) charging by alpha beta-LeuRS, indicating possible domain-domain communication within alpha beta-LeuRS. The leucine-specific domain of alpha beta-LeuRS is responsible for minihelix recognition. To ensure accurate translation of the genetic code, LeuRS functions to hydrolyze misactivated amino acids (pretransfer editing) and misaminoacylated tRNA (posttransfer editing). In contrast to tRNA(Leu), minihelix(Leu) is unable to induce posttransfer editing even upon the addition of the anticodon domain of tRNA. Therefore, the context of tRNA is crucial for the editing of mischarged products. However, the minihelix(Leu) cannot be misaminoacylated, perhaps because of the tRNA-independent pretransfer editing activity of alpha beta-LeuRS.     

A viable amino acid editing activity in the leucyl-tRNA synthetase CP1-splicing domain is not required in the yeast mitochondria

Aminoacyl-tRNA synthetases are a family of enzymes that are responsible for translating the genetic code in the first step of protein synthesis. Some aminoacyl-tRNA synthetases have editing activities to clear their mistakes and enhance fidelity. Leucyl-tRNA synthetases have a hydrolytic active site that resides in a discrete amino acid editing domain called CP1. Mutational analysis within yeast mitochondrial leucyl-tRNA synthetase showed that the enzyme has maintained an editing active site that is competent for post-transfer editing of mischarged tRNA similar to other leucyl-tRNA synthetases. These mutations that altered or abolished leucyl-tRNA synthetase editing were introduced into complementation assays. Cell viability and mitochondrial function were largely unaffected in the presence of high levels of non-leucine amino acids. In contrast, these editing-defective mutations limited cell viability in Escherichia coli. It is possible that the yeast mitochondria have evolved to tolerate lower levels of fidelity in protein synthesis or have developed alternate mechanisms to enhance discrimination of leucine from non-cognate amino acids that can be misactivated by leucyl-tRNA synthetase.     

Leucyl-tRNA synthetase from the ancestral bacterium Aquifex aeolicus contains relics of synthetase evolution

The editing reactions catalyzed by aminoacyl-tRNA synthetases are critical for the faithful protein synthesis by correcting misactivated amino acids and misaminoacylated tRNAs. We report that the isolated editing domain of leucyl-tRNA synthetase from the deep-rooted bacterium Aquifex aeolicus (alphabeta-LeuRS) catalyzes the hydrolytic editing of both mischarged tRNA(Leu) and minihelix(Leu). Within the domain, we have identified a crucial 20-amino-acid peptide that confers editing capacity when transplanted into the inactive Escherichia coli LeuRS editing domain. Likewise, fusion of the beta-subunit of alphabeta-LeuRS to the E. coli editing domain activates its editing function. These results suggest that alphabeta-LeuRS still carries the basic features from a primitive synthetase molecule. It has a remarkable capacity to transfer autonomous active modules, which is consistent with the idea that modern synthetases arose after exchange of small idiosyncratic domains. It also has a unique alphabeta-heterodimeric structure with separated catalytic and tRNA-binding sites. Such an organization supports the tRNA/synthetase coevolution theory that predicts sequential addition of tRNA and synthetase domains.     

Two conserved threonines collaborate in the Escherichia coli leucyl-tRNA synthetase amino acid editing mechanism

The aminoacyl-tRNA synthetases covalently link transfer RNAs to their cognate amino acids. Some of the tRNA synthetases have employed an editing mechanism to ensure fidelity in this first step of protein synthesis. The amino acid editing active site for Escherichia coli leucyl-tRNA synthetase resides within the CP1 domain that folds discretely from the main body of the enzyme. A portion of the editing active site is lined with conserved threonines. Previously, we identified one of these threonine residues (Thr(252)) as a critical amino acid specificity factor. On the basis of X-ray crystal structure information, two other nearby threonine residues (Thr(247) and Thr(248)) were hypothesized to interact with the editing substrate near its cleavage site. Single mutations of either of these conserved threonine residues had minimal effects on amino acid editing. However, double mutations that deleted the hydroxyl group from the neighboring threonine residues abolished amino acid editing activity. We propose that these threonine residues, which are also conserved in the homologous isoleucyl-tRNA synthetase and valyl-tRNA synthetase editing active sites, play a central role in amino acid editing. It is possible that they collaborate in stabilizing the transition state.     

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.     

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.     

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.     

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.     

Overexpressed mitochondrial leucyl-tRNA synthetase suppresses the A3243G mutation in the mitochondrial tRNA(Leu(UUR)) gene

The A3243G mutation in the human mitochondrial tRNA^Leu(UUR) gene causes a number of human diseases. This mutation reduces the level and fraction of aminoacylated tRNA^Leu(UUR) and eliminates nucleotide modification at the wobble position of the anticodon. These deficiencies are associated with mitochondrial translation defects that result in decreased levels of mitochondrial translation products and respiratory chain enzyme activities. We have suppressed the respiratory chain defects in A3243G mutant cells by overexpressing human mitochondrial leucyl-tRNA synthetase. The rates of oxygen consumption in suppressed cells were directly proportional to the levels of leucyl-tRNA synthetase. Fifteenfold higher levels of leucyl-tRNA synthetase resulted in wild-type respiratory chain function. The suppressed cells had increased steady-state levels of tRNA^Leu(UUR) and up to threefold higher steady-state levels of mitochondrial translation products, but did not have rates of protein synthesis above those in parental mutant cells. These data suggest that suppression of the A3243G mutation occurred by increasing protein stability. This suppression of a tRNA gene mutation by increasing the steady-state levels of its cognate aminoacyl-tRNA synthetase is a model for potential therapies for human pathogenic tRNA mutations.     

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.     

Yeast mitochondrial leucyl-tRNA synthetase CP1 domain has functionally diverged to accommodate RNA splicing at expense of hydrolytic editing

The yeast mitochondrial leucyl-tRNA synthetase (ymLeuRS) performs dual essential roles in group I intron splicing and protein synthesis. A specific LeuRS domain called CP1 is responsible for clearing noncognate amino acids that are misactivated during aminoacylation. The ymLeuRS CP1 domain also plays a critical role in splicing. Herein, the ymLeuRS CP1 domain was isolated from the full-length enzyme and was active in RNA splicing in vitro. Unlike its Escherichia coli LeuRS CP1 domain counterpart, it failed to significantly hydrolyze misaminoacylated tRNA(Leu). In addition and in stark contrast to the yeast domain, the editing-active E. coli LeuRS CP1 domain failed to recapitulate the splicing activity of the full-length E. coli enzyme. Although LeuRS-dependent splicing activity is rooted in an ancient adaptation for its aminoacylation activity, these results suggest that the ymLeuRS has functionally diverged to confer a robust splicing activity. This adaptation could have come at some expense to the protein's housekeeping role in aminoacylation and editing.     

Molecular modeling study of the editing active site of Escherichia coli leucyl-tRNA synthetase: two amino acid binding sites in the editing domain

Aminoacyl-tRNA synthetases (aaRSs) strictly discriminate their cognate amino acids. Some aaRSs accomplish this via proofreading and editing mechanisms. Mursinna and coworkers recently reported that substituting a highly conserved threonine (T252) with an alanine within the editing domain of Escherichia coli leucyl-tRNA synthetase (LeuRS) caused LeuRS to cleave its cognate aminoacylated leucine from tRNA(Leu) (Mursinna et al., Biochemistry 2001;40:5376-5381). To achieve atomic level insight into the role of T252 in LeuRS and the editing reaction of aaRSs, a series of molecular modeling studies including homology modeling and automated docking simulations were carried out. A 3D structure of E. coli LeuRS was constructed via homology modeling using the X-ray structure of Thermus thermophilus LeuRS as a template because the E. coli LeuRS structure is not available from X-ray or NMR studies. However, both the X-ray T. thermophilus and homology-modeled E. coli structures were used in our studies. Amino acid binding sites in the proposed editing domain, which is also called the connective polypeptide 1 (CP1) domain, were investigated by automated docking studies. The root mean square deviation (RMSD) for backbone atoms between the X-ray and homology-modeled structures was 1.18 A overall and 0.60 A for the editing (CP1) domain. Automated docking studies of a leucine ligand into the editing domain were performed for both structures: homology structure of E. coli LeuRS and X-ray structure of T. thermophilus LeuRS for comparison. The results of the docking studies suggested that there are two possible amino acid binding sites in the CP1 domain for both proteins. The first site lies near a threonine-rich region that includes the highly conserved T252 residue, which is important for amino acid discrimination. The second site is located in a flexible loop region surrounded by residues E292, A293, M295, A296, and M298. The important T252 residue is at the bottom of the first binding pocket.     

A naturally occurring nonapeptide functionally compensates for the CP1 domain of leucyl-tRNA synthetase to modulate aminoacylation activity

aaRSs (aminoacyl-tRNA synthetases) establish the rules of the genetic code by catalysing the formation of aminoacyl-tRNA. The quality control for aminoacylation is achieved by editing activity, which is usually carried out by a discrete editing domain. For LeuRS (leucyl-tRNA synthetase), the CP1 (connective peptide 1) domain is the editing domain responsible for hydrolysing mischarged tRNA. The CP1 domain is universally present in LeuRSs, except MmLeuRS (Mycoplasma mobile LeuRS). The substitute of CP1 in MmLeuRS is a nonapeptide (MmLinker). In the present study, we show that the MmLinker, which is critical for the aminoacylation activity of MmLeuRS, could confer remarkable tRNA-charging activity on the inactive CP1-deleted LeuRS from Escherichia coli (EcLeuRS) and Aquifex aeolicus (AaLeuRS). Furthermore, CP1 from EcLeuRS could functionally compensate for the MmLinker and endow MmLeuRS with post-transfer editing capability. These investigations provide a mechanistic framework for the modular construction of aaRSs and their co-ordination to achieve catalytic efficiency and fidelity. These results also show that the pre-transfer editing function of LeuRS originates from its conserved synthetic domain and shed light on future study of the mechanism.     

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.     

Attenuation of the editing activity of the Escherichia coli leucyl-tRNA synthetase allows incorporation of novel amino acids into proteins in vivo

The fidelity of translation is dependent on the specificity of the aminoacyl-tRNA synthetases (aaRSs). The aaRSs that activate the hydrophobic amino acids leucine, isoleucine, and valine employ a proofreading mechanism that hydrolyzes noncognate aminoacyl adenylates and misaminoacylated tRNAs. Discrimination between structurally similar amino acids by these AARSs is believed to operate by a double-sieve principle, wherein a separate editing domain governs hydrolysis on the basis of the size and hydrophilicity of the amino acid side chain. Leucyl-tRNA synthetase (LeuRS) relies on its editing function to correct misaminoacylation of tRNA(Leu) by isoleucine and methionine. Thr252 of Escherichia coli LeuRS has been shown previously to be important in defining the size of the editing cavity. Here we report the isolation and characterization of three LeuRS mutants with point mutations at this position (T252Y, T252L, and T252F). The proofreading activity of the synthetase is significantly impaired when an amino acid bulkier than threonine is introduced. The rate of misaminoacylation of tRNA(Leu) by isoleucine and valine increases with the increasing size of the amino acid substituent at position 252, and the noncognate amino acids norvaline and norleucine are inserted efficiently at the leucine sites of recombinant proteins under conditions of constitutive overexpression of the T252Y mutant in E. coli. In addition, the unsaturated amino acids allylglycine, homoallylglycine, homopropargylglycine, and 2-butynylalanine all support protein synthesis in E. coli hosts harboring the mutant synthetase. These results demonstrate that programmed manipulation of the editing cavity can allow in vivo incorporation of novel protein building blocks.     

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.     

An evolved aminoacyl-tRNA synthetase with atypical polysubstrate specificity

We have employed a rapid fluorescence-based screen to assess the polyspecificity of several aminoacyl-tRNA synthetases (aaRSs) against an array of unnatural amino acids. We discovered that a p-cyanophenylalanine specific aminoacyl-tRNA synthetase (pCNF-RS) has high substrate permissivity for unnatural amino acids, while maintaining its ability to discriminate against the 20 canonical amino acids. This orthogonal pCNF-RS, together with its cognate amber nonsense suppressor tRNA, is able to selectively incorporate 18 unnatural amino acids into proteins, including trifluoroketone-, alkynyl-, and halogen-substituted amino acids. In an attempt to improve our understanding of this polyspecificity, the X-ray crystal structure of the aaRS-p-cyanophenylalanine complex was determined. A comparison of this structure with those of other mutant aaRSs showed that both binding site size and other more subtle features control substrate polyspecificity.