Divergent anticodon recognition in contrasting glutamyl-tRNA synthetases

The pathogenic bacterium Helicobacter pylori utilizes two essential glutamyl-tRNA synthetases (GluRS1 and GluRS2). These two enzymes are closely related in evolution and yet they aminoacylate contrasting tRNAs. GluRS1 is a canonical discriminating GluRS (D-GluRS) that biosynthesizes Glu-tRNA(Glu) and cannot make Glu-tRNA(Gln). In contrast, GluRS2 is non-canonical as it is only essential for the production of misacylated Glu-tRNA(Gln). The co-existence and evident divergence of these two enzymes was capitalized upon to directly examine how GluRS2 acquired tRNA(Gln) specificity. One key feature that distinguishes tRNA(Glu) from tRNA(Gln) is the third position in the anticodon of each tRNA (C36 versus G36, respectively). By comparing sequence alignments of different GluRSs, including GluRS1s and GluRS2s, to the crystal structure of the Thermus thermophilus D-GluRS:tRNA(Glu) complex, a divergent pattern of conservation in enzymes that aminoacylate tRNA(Glu)versus those specific for tRNA(Gln) emerged and was experimentally validated. In particular, when an arginine conserved in discriminating GluRSs and GluRS1s was inserted into Hp GluRS2 (Glu334Arg GluRS2), the catalytic efficiency of the mutant enzyme (k(cat)/K(Mapp)) was reduced by approximately one order of magnitude towards tRNA(Gln). However, this mutation did not introduce activity towards tRNA(Glu). In contrast, disruption of a glycine that is conserved in all GluRS2s but not in other GluRSs (Gly417Thr GluRS2) generated a mutant GluRS2 with weak activity towards tRNA(Glu1). Synergy between these two mutations was observed in the double mutant (Glu334Arg/Gly417Thr GluRS2), which specifically and more robustly aminoacylates tRNA(Glu1) instead of tRNA(Gln). As GluRS1 and GluRS2 are related by an apparent gene duplication event, these results demonstrate that we can experimentally map critical evolutionary events in the emergence of new tRNA specificities.     

Crystal structure of a non-discriminating glutamyl-tRNA synthetase

Error-free protein biosynthesis is dependent on the reliable charging of each tRNA with its cognate amino acid. Many bacteria, however, lack a glutaminyl-tRNA synthetase. In these organisms, tRNA(Gln) is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase (ND-GluRS). This enzyme thus charges both tRNA(Glu) and tRNA(Gln) with glutamate. Discriminating GluRS (D-GluRS), found in some bacteria and all eukaryotes, exclusively generates Glu-tRNA(Glu). Here we present the first crystal structure of a non-discriminating GluRS from Thermosynechococcus elongatus (ND-GluRS(Tel)) in complex with glutamate at a resolution of 2.45 A. Structurally, the enzyme shares the overall architecture of the discriminating GluRS from Thermus thermophilus (D-GluRS(Tth)). We confirm experimentally that GluRS(Tel) is non-discriminating and present kinetic parameters for synthesis of Glu-tRNA(Glu) and of Glu-tRNA(Gln). Anticodons of tRNA(Glu) (34C/UUC36) and tRNA(Gln) (34C/UUG36) differ only in base 36. The pyrimidine base of C36 is specifically recognized in D-GluRS(Tth) by the residue Arg358. In ND-GluRS(Tel) this arginine residue is replaced by glycine (Gly366) presumably allowing both cytosine and the bulkier purine base G36 of tRNA(Gln) to be tolerated. Most other ND-GluRS share this structural feature, leading to relaxed substrate specificity.     

Rational design and directed evolution of a bacterial-type glutaminyl-tRNA synthetase precursor

Protein biosynthesis requires aminoacyl-transfer RNA (tRNA) synthetases to provide aminoacyl-tRNA substrates for the ribosome. Most bacteria and all archaea lack a glutaminyl-tRNA synthetase (GlnRS); instead, Gln-tRNA(Gln) is produced via an indirect pathway: a glutamyl-tRNA synthetase (GluRS) first attaches glutamate (Glu) to tRNA(Gln), and an amidotransferase converts Glu-tRNA(Gln) to Gln-tRNA(Gln). The human pathogen Helicobacter pylori encodes two GluRS enzymes, with GluRS2 specifically aminoacylating Glu onto tRNA(Gln). It was proposed that GluRS2 is evolving into a bacterial-type GlnRS. Herein, we have combined rational design and directed evolution approaches to test this hypothesis. We show that, in contrast to wild-type (WT) GlnRS2, an engineered enzyme variant (M110) with seven amino acid changes is able to rescue growth of the temperature-sensitive Escherichia coli glnS strain UT172 at its non-permissive temperature. In vitro kinetic analyses reveal that WT GluRS2 selectively acylates Glu over Gln, whereas M110 acylates Gln 4-fold more efficiently than Glu. In addition, M110 hydrolyzes adenosine triphosphate 2.5-fold faster in the presence of Glu than Gln, suggesting that an editing activity has evolved in this variant to discriminate against Glu. These data imply that GluRS2 is a few steps away from evolving into a GlnRS and provides a paradigm for studying aminoacyl-tRNA synthetase evolution using directed engineering approaches.     

Glutamyl-tRNA synthetases of Bacillus subtilis 168T and of Bacillus stearothermophilus. Cloning and sequencing of the gltX genes and comparison with other aminoacyl-tRNA synthetases

The glutamyl-tRNA synthetase (GluRS) of Bacillus subtilis 168T aminoacylates with glutamate its homologous tRNA(Glu) and tRNA(Gln) in vivo and Escherichia coli tRNA(1Gln) in vitro (Lapointe, J., Duplain, L., and Proulx, M. (1986) J. Bacteriol. 165, 88-93). The gltX gene encoding this enzyme was cloned and sequenced. It encodes a protein of 483 amino acids with a Mr of 55,671. Alignment of the amino acid sequences of four bacterial GluRSs (from B. subtilis, Bacillus stearothermophilus, E. coli, and Rhizobium meliloti) gives 20% identity and reveals the presence of several short highly conserved motifs in the first two thirds of these proteins. Conserved motifs are found at corresponding positions in several other aminoacyl-tRNA synthetases. The only sequence similarity between the GluRSs of these Bacillus species and the E. coli glutaminyl-tRNA synthetase (GlnRS), which has no counterpart in the E. coli GluRS, is in a segment of 30 amino acids in the last third of these synthetases. In the three-dimensional structure of the E. coli tRNA(Gln).GlnRS.ATP complex, this conserved peptide is near the anticodon of tRNA(Gln) (Rould, M. A., Perona, J. J., S?ll, D., and Steitz, T. A. (1989) Science 246, 1135-1142), suggesting that this region is involved in the specific interactions between these enzymes and the anticodon regions of their tRNA substrates.     

Recognition of tRNAGln by Helicobacter pylori GluRS2—a tRNAGln-specific glutamyl-tRNA synthetase

Accurate aminoacylation of tRNAs by the aminoacyl-tRNA synthetases (aaRSs) plays a critical role in protein translation. However, some of the aaRSs are missing in many microorganisms. Helicobacter pylori does not have a glutaminyl-tRNA synthetase (GlnRS) but has two divergent glutamyl-tRNA synthetases: GluRS1 and GluRS2. Like a canonical GluRS, GluRS1 aminoacylates tRNA^Glu1 and tRNA^Glu2. In contrast, GluRS2 only misacylates tRNA^Gln to form Glu-tRNA^Gln. It is not clear how GluRS2 achieves specific recognition of tRNA^Gln while rejecting the two H. pylori tRNA^Glu isoacceptors. Here, we show that GluRS2 recognizes major identity elements clustered in the tRNA^Gln acceptor stem. Mutations in the tRNA anticodon or at the discriminator base had little to no impact on enzyme specificity and activity.     

The zinc-binding site of a class I aminoacyl-tRNA synthetase is a SWIM domain that modulates amino acid binding via the tRNA acceptor arm.

In its tRNA acceptor end binding domain, the glutamyl-tRNA synthetase (GluRS) of Escherichia coli contains one atom of zinc that holds the extremities of a segment (Cys98-x-Cys100-x24-Cys125-x-His127) homologous to the Escherichia coli glutaminyl-tRNA synthetase (GlnRS) loop where a leucine residue stabilizes the peeled-back conformation of tRNAGln acceptor end. We report here that the GluRS zinc-binding region belongs to the novel SWIM domain family characterized by the signature C-x-C-xn-C-x-H (n = 6-25), and predicted to interact with DNA or proteins. In the presence of tRNAGlu, the GluRS C100Y variant has a lower affinity for l-glutamate than the wild-type enzyme, with Km and Kd values increased 12- and 20-fold, respectively. On the other hand, in the absence of tRNAGlu, glutamate binds with the same affinity to the C100Y variant and to wild-type GluRS. In the context of the close structural and mechanistic similarities between GluRS and GlnRS, these results indicate that the GluRS SWIM domain modulates glutamate binding to the active site via its interaction with the tRNAGlu acceptor arm. Phylogenetic analyses indicate that ancestral GluRSs had a strong zinc-binding site in their SWIM domain. Considering that all GluRSs require a cognate tRNA to activate glutamate, and that some of them have different or no putative zinc-binding residues in the corresponding positions, the properties of the C100Y variant suggest that the GluRS SWIM domains evolved to position correctly the tRNA acceptor end in the active site, thereby contributing to the formation of the glutamate binding site.     

The identity determinants required for the discrimination between tRNAGlu and tRNAAsp by glutamyl-tRNA synthetase from Escherichia coli.

We previously elucidated the major determinant set for Escherichia coli tRNAGlu identity (U34, U35, C36, A37, G1*C72, U2*A71, U11*A24, U13*G22**Alpha46, and Delta47) and showed that the set is sufficient to switch the identity of tRNAGln to Glu [Sekine, S., Nureki, O., Sakamoto, K., Niimi, T., Tateno, M., Go, M., Kohno, T., Brisson, A., Lapointe, J. & Yokoyama, S. (1996) J. Mol. Biol. 256, 685-700]. In the present study, we attempted to switch the identity of tRNAAsp, which has a sequence similar to that of tRNAGlu, and consequently possesses many nucleotide residues corresponding to the Glu identity determinants (U35, C36, A37, G1*C72, and U11*A24). A simple transplantation of the rest of the major determinants (U34, U2*A71, U13*G22**Alpha46, and Delta47) to the framework of tRNAAsp did not result in a sufficient switch of the tRNAAsp identity to Glu. To confer an optimal glutamate accepting activity to tRNAAsp, two other elements, C4*G69 in the middle of the acceptor stem and C12*G23**C9 in the augmented D helix, were required. Consistently, the two base pairs, C4*G69 and C12*G23, in tRNAGlu had been shown to exist in the interface with glutamyl-tRNA synthetase (GluRS) by phosphate-group footprinting. We also found the two elements in the framework of tRNAGln, and determined that their contributions successfully changed the identity of tRNAGln to Glu in the previous study. By the identity-determinant set (C4*G69 and C12*G23**C9 in addition to U34, U35, C36, A37, G1*C72, U2*A71, U11*A24, U13*G22**Alpha46, and Delta47) the activity of GluRS was optimized and efficient discrimination from the noncognate tRNAs was achieved.     

Anticodon recognition and discrimination by the alpha-helix cage domain of class I lysyl-tRNA synthetase

Aminoacyl-tRNA synthetases are normally found in one of two mutually exclusive structural classes, the only known exception being lysyl-tRNA synthetase which exists in both classes I (LysRS1) and II (LysRS2). Differences in tRNA acceptor stem recognition between LysRS1 and LysRS2 do not drastically impact cellular aminoacylation levels, focusing attention on the mechanism of tRNA anticodon recognition by LysRS1. On the basis of structure-based sequence alignments, seven tRNALys anticodon variants and seven LysRS1 anticodon binding site variants were selected for analysis of the Pyrococcus horikoshii LysRS1-tRNALys docking model. LysRS1 specifically recognized the bases at positions 35 and 36, but not that at position 34. Aromatic residues form stacking interactions with U34 and U35, and aminoacylation kinetics also identified direct interactions between Arg502 and both U35 and U36. Tyr491 was also found to interact with U36, and the Y491E variant exhibited significant improvement compared to the wild type in aminoacylation of a tRNALysUUG mutant. Refinement of the LysRS1-tRNALys docking model based upon these data suggested that anticodon recognition by LysRS1 relies on considerably fewer interactions than that by LysRS2, providing a structural basis for the more significant role of the anticodon in tRNA recognition by the class II enzyme. To date, only glutamyl-tRNA synthetase (GluRS) has been found to contain an alpha-helix cage anticodon binding domain homologous to that of LysRS1, and these data now suggest that specificity for the anticodon of tRNALys could have been acquired through relatively few changes to the corresponding domain of an ancestral GluRS enzyme.     

A Functional Loop Spanning Distant Domains of Glutaminyl-tRNA Synthetase Also Stabilizes a Molten Globule State

Molten globule and other disordered states of proteins are now known to play important roles in many cellular processes. From equilibrium unfolding studies of two paralogous proteins and their variants, glutaminyl-tRNA synthetase (GlnRS) and two of its variants [glutamyl-tRNA synthetase (GluRS) and its isolated domains, and a GluRS-GlnRS chimera], we demonstrate that only GlnRS forms a molten globule-like intermediate at low urea concentrations. We demonstrated that a loop in the GlnRS C-terminal anticodon binding domain that promotes communication with the N-terminal domain and indirectly modulates amino acid binding is also responsible for stabilization of the molten globule state. This loop was inserted into GluRS in the eukaryotic branch after the archaea-eukarya split, right around the time when GlnRS evolved. Because of the structural and functional importance of the loop, it is proposed that the insertion of the loop into a putative ancestral GluRS in eukaryotes produced a catalytically active molten globule state. Because of their enhanced dynamic nature, catalytically active molten globules are likely to possess broad substrate specificity. It is further proposed that the putative broader substrate specificity allowed the catalytically active molten globule to accept glutamine in addition to glutamic acid, leading to the evolution of GlnRS.     

Structural and functional consequences of mutating a proteobacteria-specific surface residue in the catalytic domain of Escherichia coli GluRS

Nucleotides whose mutations seriously affect glutamylation efficiency are experimentally known for Escherichia coli tRNA(Glu). However, not much is known about functional hotspots on the complementary enzyme, glutamyl-tRNA synthetase (GluRS). From structural and functional studies on an Arg266Leu mutant of E. coli GluRS, we demonstrate that Arg266 is essential for efficient glutamylation of tRNA(Glu). Consistent with this result, we found that Arg266 is a conserved signature of proteobacterial GluRS. In contrast, most non-proteobacterial GluRS contain Leu, and never Arg, at this position. Our results imply a unique strategy of glutamylation of tRNA(Glu) in proteobacteria under phylum-specific evolutionary compulsions.     

Conformation change of tRNAGlu in the complex with glutamyl-tRNA synthetase is required for the specific binding of L-glutamate

The binding of Thermus thermophilus glutamyl-tRNA synthetase (GluRS) with T. thermophilus tRNAGlu, Escherichia coli tRNAGlu, and amino acids was studied by fluorescence measurements. In the absence of tRNAGlu, GluRS binds with D-glutamate as well as L-glutamate. However, in the presence of E. coli tRNAGlu, GluRS binds specifically with L-glutamate. The KCl effects on the Michaelis constants (Km) for tRNAGlu, L-glutamate, and ATP were studied for the aminoacylation of the homologous tRNAGlu and heterologous tRNAGlu species. As the KCl concentration is raised from 0 to 100 mM, the Km value for L-glutamate in the heterologous system is remarkably increased whereas the Km value for L-glutamate in the homologous system is only slightly increased. The circular dichroism analyses were made mainly of the bands due to the 2-thiouridine derivatives of tRNAGlu in the complex. The conformation change of T. thermophilus tRNAGlu upon complex formation with GluRS is not affected by addition of KCl. In contrast, the heterologous tRNAGlu X GluRS complex is in an equilibrium of two forms that depends on KCl concentration. The predominant form at low KCl concentration is closely related to the small Km value for L-glutamate. In this form of the complex, the conformation of tRNAGlu is appreciably different from that of free molecule. Accordingly, such a conformation change of tRNAGlu in the complex with GluRS is required for the specific binding of L-glutamate as the substrate.     

Growth inhibition of Escherichia coli during heterologous expression of Bacillus subtilis glutamyl-tRNA synthetase that catalyzes the formation of mischarged glutamyl-tRNA1 Gln

It is known that Bacillus subtilis glutamyl-tRNA synthetase (GluRS) mischarges E. coli tRNA1 Gln with glutamate in vitro. It has also been established that the expression of B. subtilis GluRS in Escherichia coli results in the death of the host cell. To ascertain whether E. coli growth inhibition caused by B. subtilis GluRS synthesis is a consequence of Glu-tRNA1 Ghn formation, we constructed an in vivo test system, in which B. subtilis GluRS gene expression is controlled by IPTG. Such a system permits the investigation of factors affecting E. coli growth. Expression of E. coli glutaminyl-tRNA synthetase (GlnRS) also ameliorated growth inhibition, presumably by competitively preventing tRNA1 Gln misacylation. However, when amounts of up to 10 mM L-glutamine, the cognate amino acid for acylation of tRNA1 Gln, were added to the growth medium, cell growth was unaffected. Overexpression of the B. subtilis gatCAB gene encoding Glu-tRNAGln amidotransferase (Glu-AdT) rescued cells from toxic effects caused by the formation of the mischarging GluRS. This result indicates that B. subtilis Glu-AdT recognizes the mischarged E. coli GlutRNA1 Gln, and converts it to the cognate Gln-tRNA1 Gln species. B. subtilis GluRS-dependent Glu-tRNA1 Gln formation may cause growth inhibition in the transformed E. coli strain, possibly due to abnormal protein synthesis.     

A chimaeric glutamyl:glutaminyl-tRNA synthetase: implications for evolution

aaRSs (aminoacyl-tRNA synthetases) are multi-domain proteins that have evolved by domain acquisition. The anti-codon binding domain was added to the more ancient catalytic domain during aaRS evolution. Unlike in eukaryotes, the anti-codon binding domains of GluRS (glutamyl-tRNA synthetase) and GlnRS (glutaminyl-tRNA synthetase) in bacteria are structurally distinct. This originates from the unique evolutionary history of GlnRSs. Starting from the catalytic domain, eukaryotic GluRS evolved by acquiring the archaea/eukaryote-specific anti-codon binding domain after branching away from the eubacteria family. Subsequently, eukaryotic GlnRS evolved from GluRS by gene duplication and horizontally transferred to bacteria. In order to study the properties of the putative ancestral GluRS in eukaryotes, formed immediately after acquiring the anti-codon binding domain, we have designed and constructed a chimaeric protein, cGluGlnRS, consisting of the catalytic domain, Ec GluRS (Escherichia coli GluRS), and the anti-codon binding domain of EcGlnRS (E. coli GlnRS). In contrast to the isolated EcN-GluRS, cGluGlnRS showed detectable activity of glutamylation of E. coli tRNA(glu) and was capable of complementing an E. coli ts (temperature-sensitive)-GluRS strain at non-permissive temperatures. Both cGluGlnRS and EcN-GluRS were found to bind E. coli tRNA(glu) with native EcGluRS-like affinity, suggesting that the anticodon-binding domain in cGluGlnRS enhances k(cat) for glutamylation. This was further confirmed from similar experiments with a chimaera between EcN-GluRS and the substrate-binding domain of EcDnaK (E. coli DnaK). We also show that an extended loop, present in the anticodon-binding domains of GlnRSs, is absent in archaeal GluRS, suggesting that the loop was a later addition, generating additional anti-codon discrimination capability in GlnRS as it evolved from GluRS in eukaryotes.     

In vivo formation of glutamyl-tRNA(Gln) in Escherichia coli by heterologous glutamyl-tRNA synthetases

Two types of glutamyl-tRNA synthetase exist: the discriminating enzyme (D-GluRS) forms only Glu-tRNA(Glu), while the non-discriminating one (ND-GluRS) also synthesizes Glu-tRNA(Gln), a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). Testing the capacity to complement a thermosensitive E. coli gltX mutant and to suppress an E. coli trpA49 missense mutant we examined the properties of heterologous gltX genes. We demonstrate that while Acidithiobacillus ferrooxidans GluRS1 and Bacillus subtilis Q373R GluRS form Glu-tRNA(Glu), A. ferrooxidans and Helicobacter pylori GluRS2 form Glu-tRNA(Gln) in E. coli in vivo.     

Crystal structure of glutamyl-queuosine tRNAAsp synthetase complexed with L-glutamate: structural elements mediating tRNA-independent activation of glutamate and glutamylation of tRNAAsp anticodon

Glutamyl-queuosine tRNA^Asp synthetase (Glu-Q-RS) from Escherichia coli is a paralog of the catalytic core of glutamyl-tRNA synthetase (GluRS) that catalyzes glutamylation of queuosine in the wobble position of tRNA^Asp. Despite important structural similarities, Glu-Q-RS and GluRS diverge strongly by their functional properties. The only feature common to both enzymes consists in the activation of Glu to form Glu-AMP, the intermediate of transfer RNA (tRNA) aminoacylation. However, both enzymes differ by the mechanism of selection of the cognate amino acid and by the mechanism of its activation. Whereas GluRS selects l-Glu and activates it only in the presence of the cognate tRNA^Glu, Glu-Q-RS forms Glu-AMP in the absence of tRNA. Moreover, while GluRS transfers the activated Glu to the 3′ accepting end of the cognate tRNA^Glu, Glu-Q-RS transfers the activated Glu to Q34 located in the anticodon loop of the noncognate tRNA^Asp. In order to gain insight into the structural elements leading to distinct mechanisms of amino acid activation, we solved the three-dimensional structure of Glu-Q-RS complexed to Glu and compared it to the structure of the GluRS·Glu complex. Comparison of the catalytic site of Glu-Q-RS with that of GluRS, combined with binding experiments of amino acids, shows that a restricted number of residues determine distinct catalytic properties of amino acid recognition and activation by the two enzymes. Furthermore, to explore the structural basis of the distinct aminoacylation properties of the two enzymes and to understand why Glu-Q-RS glutamylates only tRNA^Asp among the tRNAs possessing queuosine in position 34, we performed a tRNA mutational analysis to search for the elements of tRNA^Asp that determine recognition by Glu-Q-RS. The analyses made on tRNA^Asp and tRNA^Asn show that the presence of a C in position 38 is crucial for glutamylation of Q34. The results are discussed in the context of the evolution and adaptation of the tRNA glutamylation system.     

Seven, eight and nine-membered anticodon loop mutants of tRNA(2Arg) which cause +1 frameshifting. Tolerance of DHU arm and other secondary mutations.

The mutant tRNA(2Arg) encoded by the genetically-selected frameshift suppressor, sufT621, inserts arginine and causes a +1 reading-frame shift at the proline codon, CCG(U). There is an extra base, G36.1, in argV beta, one of the four identical genes for tRNA(2Arg) in the position between bases 36 and 37, corresponding to the 3' side of the anticodon. The new four-base anticodon, predicted from DNA sequencing to be 3' GGCA 5', is complementary to the four-base codon CCGU. Quadruplet translocation promoted by mutant argV does not require perfect complementarity between the codon and the anticodon since synthetic genes encoding derivatives of tRNA(2Arg) and tRNA(1Pro), with four-base anticodons complementary to three out of the four bases of CCGU, were also shown to be capable of frameshifting. Two other mutants of argV, inferred to have normal-size, seven-base anticodon loops, were also found to be capable of four-base-decoding demonstrating that quadruplet translocation promoted by mutant argV does not require an enlarged anticodon loop. Other alleles of argV, predicted to have nine bases in the anticodon loop, were also found to cause frameshifting. The DNA sequence of two of these showed in addition, either a deletion of G24, or a ten-base duplication in the region corresponding to the TFC arm. A general finding is that mutations in the DHU arm of tRNA(2Arg) are compatible with, and in one case necessary for, frameshifting.     

Nonsense and missense translational suppression in plant cells mediated by tRNALys

A nuclear tRNA^Lys gene from Arabidopsis thaliana was cloned and mutated so as to express tRNAs with altered anticodons which bind to a UAG nonsense (amber) codon and to the Arg (AGG), Asn (AAC,AAT), Gln (CAG) or Glu (GAG) codons. Concomitantly, a codon in the firefly luciferase gene for a functionally important Lys was altered to an amber codon, or to Arg, Asn, Gln, Glu, Thr and Trp codons, so as to construct reporter genes reliant upon incorporation of Lys. The altered tRNA^Lys and luciferase genes were introduced into Nicotiana benthamiana protoplasts and expression of the mutated tRNAs was verified by translational suppression of the mutant firefly luciferase genes. Expression of the amber suppressor tRNA _CUA ^Lys from non-replicative vectors promoted 10–40% suppression of the luciferase nonsense reporters while expression of the amber and missense tRNA^Lys suppressor genes from a geminivirus vector capable of replication promoted 30–80% suppression of the luciferase nonsense reporter and up to 10% suppression of the luciferase missense reporters with Arg, Asn, Gln and Glu codons.     

An Extensive Study of Mutation and Selection on the Wobble Nucleotide in tRNA Anticodons in Fungal Mitochondrial Genomes

Two alternative hypotheses aim to predict the wobble nucleotide of tRNA anticodons in mitochondrion. The codon-anticodon adaptation hypothesis predicts that the wobble nucleotide of tRNA anticodon should evolve toward maximizing the Watson-Crick base pairing with the most frequently used codon within each synonymous codon family. In contrast, the wobble versatility hypothesis argues that the nucleotide at the wobble site should be occupied by a nucleotide most versatile in wobble pairing, i.e., the wobble site of the tRNA anticodon should be G for NNY codon families and U for NNR and NNN codon families (where Y stands for C or U, R for A or G, and N for any nucleotide). We examined codon usage and anticodon wobble sites in 36 fungal genomes to evaluate these two alternative hypotheses and identify exceptional cases that deserve new explanations. While the wobble versatility hypothesis is generally supported, there are interesting exceptions involving tRNA(Arg) translating the CGN codon family, tRNA(Trp) translating the UGR codon family, and tRNA(Met) translating the AUR codon family. Our results suggest that the potential to suppress stop codons, the historical inertia, and the conflict between translation initiation and elongation can all contribute to determining the wobble nucleotide of tRNA anticodons.     

Gene Descent, Duplication, and Horizontal Transfer in the Evolution of Glutamyl- and Glutaminyl-tRNA Synthetases

In translation, separate aminoacyl-tRNA synthetases attach the 20 different amino acids to their cognate tRNAs, with the exception of glutamine. Eukaryotes and some bacteria employ a specific glutaminyl-tRNA synthetase (GlnRS) which other Bacteria, the Archaea (archaebacteria), and organelles apparently lack. Instead, tRNA^Gln is initially acylated with glutamate by glutamyl-tRNA synthetase (GluRS), then the glutamate moiety is transamidated to glutamine. Lamour et al. [(1994) Proc Natl Acad Sci USA 91:8670–8674] suggested that an early duplication of the GluRS gene in eukaryotes gave rise to the gene for GlnRS—a copy of which was subsequently transferred to proteobacteria. However, questions remain about the occurrence of GlnRS genes among the Eucarya (eukaryotes) outside of the ``crown' taxa (animals, fungi, and plants), the distribution of GlnRS genes in the Bacteria, and their evolutionary relationships to genes from the Archaea. Here, we show that GlnRS occurs in the most deeply branching eukaryotes and that putative GluRS genes from the Archaea are more closely related to GlnRS and GluRS genes of the Eucarya than to those of Bacteria. There is still no evidence for the existence of GlnRS in the Archaea. We propose that the last common ancestor to contemporary cells, or cenancestor, used transamidation to synthesize Gln-tRNA^Gln and that both the Bacteria and the Archaea retained this pathway, while eukaryotes developed a specific GlnRS gene through the duplication of an existing GluRS gene. In the Bacteria, GlnRS genes have been identified in a total of 10 species from three highly diverse taxonomic groups: Thermus/Deinococcus, Proteobacteria γ/β subdivision, and Bacteroides/Cytophaga/Flexibacter. Although all bacterial GlnRS form a monophyletic group, the broad phyletic distribution of this tRNA synthetase suggests that multiple gene transfers from eukaryotes to bacteria occurred shortly after the Archaea–eukaryote divergence.     

Coevolution of Specificity Determinants in Eukaryotic Glutamyl- and Glutaminyl-tRNA Synthetases

The glutaminyl-tRNA synthetase (GlnRS) enzyme, which pairs glutamine with tRNA^Gln for protein synthesis, evolved by gene duplication in early eukaryotes from a nondiscriminating glutamyl-tRNA synthetase (GluRS) that aminoacylates both tRNA^Gln and tRNA^Glu with glutamate. This ancient GluRS also separately differentiated to exclude tRNA^Gln as a substrate, and the resulting discriminating GluRS and GlnRS further acquired additional protein domains assisting function in cis (the GlnRS N-terminal Yqey domain) or in trans (the Arc1p protein associating with GluRS). These added domains are absent in contemporary bacterial GlnRS and GluRS. Here, using Saccharomyces cerevisiae enzymes as models, we find that the eukaryote-specific protein domains substantially influence amino acid binding, tRNA binding and aminoacylation efficiency, but they play no role in either specific nucleotide readout or discrimination against noncognate tRNA. Eukaryotic tRNA^Gln and tRNA^Glu recognition determinants are found in equivalent positions and are mutually exclusive to a significant degree, with key nucleotides located adjacent to portions of the protein structure that differentiated during the evolution of archaeal nondiscriminating GluRS to GlnRS. These findings provide important corroboration for the evolutionary model and suggest that the added eukaryotic domains arose in response to distinctive selective pressures associated with the greater complexity of the eukaryotic translational apparatus. We also find that the affinity of GluRS for glutamate is significantly increased when Arc1p is not associated with the enzyme. This is consistent with the lower concentration of intracellular glutamate and the dissociation of the Arc1p:GluRS complex upon the diauxic shift to respiratory conditions.