A novel homoplasmic mt-tRNAGlu m.14701C>T variant presenting with a partially reversible infantile respiratory chain deficiency.

BACKGROUND: Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial disorder associated with variable penetrance and partial to full remission of symptoms. OBJECTIVE: To describe features of maternally related individuals with a novel variant associated with RIRCD. MATERIALS AND METHODS: Nine maternally related individuals aged 23 months to 64 years are described through physical examinations, muscle biopsies, histochemical and biochemical analyses, genome sequencing, and cerebral imaging. RESULTS: A homoplasmic mitochondrial transfer ribonucleic acid for glutamic acid (mt-tRNAGlu) m.14701C>T variant was identified in eight tested individuals out of nine maternally related individuals. Two individuals presented with hypotonia, muscle weakness, feeding difficulties and lactic acidosis at age 3-4 months, and improvement around age 15-23 months with mild residual symptoms at last examination. One individual with less severe symptoms had unknown age at onset and improved around age 4-5 years. Five individuals developed lipoma on the upper back, and one adult individual developed ataxia, while one was unaffected. CONCLUSIONS: We have identified a novel homoplasmic mt-tRNAGlu m.14701C>T variant presenting with phenotypic and paraclinical features associated with RIRCD as well as ataxia and lipomas, which to our knowledge are new features associated to RIRCD.

A novel 3'-tRNAGlu-derived fragment acts as a tumor suppressor in breast cancer by targeting nucleolin.

tRNA-derived fragments (tRFs) have been defined as a novel class of small noncoding RNAs. tRFs have been reported to be deregulated in cancer, but their biologic function remains to be fully understood. We have identified a new tRF (named tRF3E), derived from mature tRNAGlu, that is specifically expressed in healthy mammary glands but not in breast cancer (BC). Consistently, tRF3E levels significantly decrease in the blood of patients with epidermal growth factor receptor 2 (HER2)-positive BC reflecting tumor status (control > early cancer > metastatic cancer). tRF3E down-regulation was recapitulated in Δ16HER2 transgenic mice, representing a BC preclinical model. Pulldown assays, used to search for proteins capable to selectively bind tRF3E, have shown that this tRF specifically interacts with nucleolin (NCL), an RNA-binding protein overexpressed in BC and able to repress the translation of p53 mRNA. The binding properties of NCL-tRF3E complex, predicted in silico and analyzed by EMSA assays, are congruent with a competitive displacement of p53 mRNA by tRF3E, leading to an increased p53 expression and consequently to a modulation of cancer cell growth. Here, we provide evidence that tRF3E plays an important role in the pathogenesis of BC displaying tumor-suppressor functions through a NCL-mediated mechanism.-Falconi, M., Giangrossi, M., Elexpuru Zabaleta, M., Wang, J., Gambini, V., Tilio, M., Bencardino, D., Occhipinti, S., Belletti, B., Laudadio, E., Galeazzi, R., Marchini, C., Amici, A. A novel 3'-tRNAGlu-derived fragment acts as a tumor suppressor in breast cancer by targeting nucleolin.

Logical engineering of D-arm and T-stem of tRNA that enhances d-amino acid incorporation.

A bacterial translation factor EF-P alleviates ribosomal stalling caused by polyproline sequence by accelerating Pro-Pro formation. EF-P recognizes a specific D-arm motif found in tRNAPro isoacceptors, 9-nt D-loop closed by a stable D-stem sequence, for Pro-selective peptidyl-transfer acceleration. It is also known that the T-stem sequence on aminoacyl-tRNAs modulates strength of the interaction with EF-Tu, giving enhanced incorporation of non-proteinogenic amino acids such as some N-methyl amino acids. Based on the above knowledge, we logically engineered tRNA's D-arm and T-stem sequences to investigate a series of tRNAs for the improvement of consecutive incorporation of d-amino acids and an α, α-disubstituted amino acid. We have devised a chimera of tRNAPro1 and tRNAGluE2, referred to as tRNAPro1E2, in which T-stem of tRNAGluE2 was engineered into tRNAPro1. The combination of EF-P with tRNAPro1E2NNN pre-charged with d-Phe, d-Ser, d-Ala, and/or d-Cys has drastically enhanced expression level of not only linear peptides but also a thioether-macrocyclic peptide consisting of the four consecutive d-amino acids over the previous method using orthogonal tRNAs.

A Deafness- and Diabetes-associated tRNA Mutation Causes Deficient Pseudouridinylation at Position 55 in tRNAGlu and Mitochondrial Dysfunction.

Several mitochondrial tRNA mutations have been associated with maternally inherited diabetes and deafness. However, the pathophysiology of these tRNA mutations remains poorly understood. In this report, we identified the novel homoplasmic 14692A→G mutation in the mitochondrial tRNAGlu gene among three Han Chinese families with maternally inherited diabetes and deafness. The m.14692A→G mutation affected a highly conserved uridine at position 55 of the TΨC loop of tRNAGlu The uridine is modified to pseudouridine (Ψ55), which plays an important role in the structure and function of this tRNA. Using lymphoblastoid cell lines derived from a Chinese family, we demonstrated that the m.14692A→G mutation caused loss of Ψ55 modification and increased angiogenin-mediated endonucleolytic cleavage in mutant tRNAGlu The destabilization of base-pairing (18A-Ψ55) caused by the m.14692A→G mutation perturbed the conformation and stability of tRNAGlu An approximately 65% decrease in the steady-state level of tRNAGlu was observed in mutant cells compared with control cells. A failure in tRNAGlu metabolism impaired mitochondrial translation, especially for polypeptides with a high proportion of glutamic acid codons such as ND1, ND6, and CO2 in mutant cells. An impairment of mitochondrial translation caused defective respiratory capacity, especially reducing the activities of complexes I and IV. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These mitochondrial dysfunctions caused an increasing production of reactive oxygen species in the mutant cells. Our findings may provide new insights into the pathophysiology of maternally inherited diabetes and deafness, which is primarily manifested by the deficient nucleotide modification of mitochondrial tRNAGlu.

Molecular mechanism of bacterial persistence by HipA.

HipA of Escherichia coli is a eukaryote-like serine-threonine kinase that inhibits cell growth and induces persistence (multidrug tolerance). Previously, it was proposed that HipA inhibits cell growth by the phosphorylation of the essential translation factor EF-Tu. Here, we provide evidence that EF-Tu is not a target of HipA. Instead, a genetic screen reveals that the overexpression of glutamyl-tRNA synthetase (GltX) suppresses the toxicity of HipA. We show that HipA phosphorylates conserved Ser(239) near the active center of GltX and inhibits aminoacylation, a unique example of an aminoacyl-tRNA synthetase being inhibited by a toxin encoded by a toxin-antitoxin locus. HipA only phosphorylates tRNA(Glu)-bound GltX, which is consistent with the earlier finding that the regulatory motif containing Ser(239) changes configuration upon tRNA binding. These results indicate that HipA mediates persistence by the generation of "hungry" codons at the ribosomal A site that trigger the synthesis of (p)ppGpp, a hypothesis that we verify experimentally.

Early-onset cataracts, spastic paraparesis, and ataxia caused by a novel mitochondrial tRNAGlu (MT-TE) gene mutation causing severe complex I deficiency: a clinical, molecular, and neuropathologic study.

Mitochondrial respiratory chain disease is associated with a spectrum of clinical presentations and considerable genetic heterogeneity. Here we report molecular genetic and neuropathologic findings from an adult with an unusual manifestation of mitochondrial DNA disease. Clinical features included early-onset cataracts, ataxia, and progressive paraparesis, with sequencing revealing the presence of a novel de novo m.14685G>A mitochondrial tRNA(Glu) (MT-TE) gene mutation. Muscle biopsy showed that 13% and 34% of muscle fibers lacked cytochrome c oxidase activity and complex I subunit expression, respectively. Biochemical studies confirmed a marked decrease in complex I activity. Neuropathologic investigation revealed a large cystic lesion affecting the left putamen, caudate nucleus, and internal capsule, with evidence of marked microvacuolation, neuron loss, perivascular lacunae, and blood vessel mineralization. The internal capsule showed focal axonal loss, whereas brainstem and spinal cord showed descending anterograde degeneration in medullary pyramids and corticospinal tracts. In agreement with muscle biopsy findings, reduced complex I immunoreactivity was detected in the remaining neuronal populations, particularly in the basal ganglia and cerebellum, correlating with the neurologic dysfunction exhibited by the patient. This study emphasizes the importance of molecular genetic and postmortem neuropathologic analyses for furthering our understanding of underlying mechanisms of mitochondrial disorders.

A novel mitochondrial tRNAGlu (MTTE) gene mutation causing chronic progressive external ophthalmoplegia at low levels of heteroplasmy in muscle.

Mitochondrial respiratory chain defects are associated with diverse clinical phenotypes in both adults and children, and may be caused by mutations in either nuclear or mitochondrial DNA (mtDNA). We report the molecular genetic investigations of a patient with chronic progressive external ophthalmoplegia (CPEO) and myopathy where muscle biopsies taken 11 years apart revealed a progressive increase in the proportion of cytochrome c oxidase (COX)-deficient fibres. Mitochondrial genetic analysis of the early biopsy had seemingly excluded both mtDNA rearrangements and mtDNA point mutations. Sequencing mtDNA from individual COX-deficient muscle fibres in the second biopsy, however, identified an unreported m.14723T>C substitution within the mitochondrial tRNAGlu (MTTE) gene, which fulfilled all canonical criteria for pathogenicity. The m.14723T>C mutation was absent from several tissues, including muscle, from maternal relatives suggesting a de novo event, whilst quantitative analysis of the first muscle biopsy confirmed a very low level of the mutation (7% mutated mtDNA), highlighting a potential problem whereby pathogenic mtDNA mutations may remain undetected using established screening methodologies.

ND6 gene "lost" and found: evolution of mitochondrial gene rearrangement in Antarctic notothenioids.

Evolution of Antarctic notothenioids in the frigid and oxygen-rich Southern Ocean had led to remarkable genomic changes, most notably the gain of novel antifreeze glycoproteins and the loss of oxygen-binding hemoproteins in the icefish family. Recently, the mitochondrial (mt) NADH dehydrogenase subunit 6 (ND6) gene and the adjacent transfer RNA(Glu) (tRNA(Glu)) were also reportedly lost. ND6 protein is crucial for the assembly and function of Complex I of the mt electron transport chain that produces adenosine triphosphate (ATP) essential for life; thus, ND6 absence would be irreconcilable with Antarctic notothenioids being thriving species. Here we report our discovery that the ND6 gene and tRNA(Glu) were not lost but had been translocated to the control region (CR) from their canonical location between ND5 and cytochrome b genes. We characterized the CR and adjacent sequences of 22 notothenioid species representing all eight families of Notothenioidei to elucidate the mechanism and evolutionary history of this mtDNA rearrangement. Species of the three basal non-Antarctic families have the canonical vertebrate mt gene order, whereas species of all five Antarctic families have a rearranged CR bearing the embedded ND6 (ND6(CR)) and tRNA(Glu), with additional copies of tRNA(Thr), tRNA(Pro), and noncoding region in various lineages. We hypothesized that an initial duplication of the canonical mt region from ND6 through CR occurred in the common ancestor to the Antarctic clade, and we deduced the succession of loss or modification of the duplicated region leading to the extant patterns of mt DNA reorganization that is consistent with notothenioid evolutionary history. We verified that the ND6(CR) gene in Antarctic notothenioids is transcribed and therefore functional. However, ND6(CR)-encoded protein sequences differ substantially from basal non-Antarctic notothenioid ND6, and we detected lineage-specific positive selection on the branch leading to the Antarctic clade of ND6(CR) under the branch-site model. Collectively, the novel mt ND6(CR) genotype of the Antarctic radiation represents another major molecular change in Antarctic notothenioid evolution and may reflect an adaptive change conducive to the functioning of the protein (Complex I) machinery of mt respiration in the polar environment, driven by the advent of freezing, oxygen-rich conditions in the Southern Ocean.

Thio-modification of yeast cytosolic tRNA requires a ubiquitin-related system that resembles bacterial sulfur transfer systems.

The wobble uridine in yeast cytosolic tRNA(Lys2)(UUU) and tRNA(Glu3)(UUC) undergoes a thio-modification at the second position (s(2) modification) and a methoxycarbonylmethyl modification at the fifth position (mcm(5) modification). We previously demonstrated that the cytosolic and mitochondrial iron-sulfur (Fe/S) cluster assembly machineries termed CIA and ISC, including a cysteine desulfurase called Nfs1, were essential for the s(2) modification. However, the cytosolic component that directly participates in this process remains unclear. We found that ubiquitin-like protein Urm1 and ubiquitin-activating enzyme-like protein Uba4, as well as Tuc1 and Tuc2, were strictly required for the s(2) modification. The carboxyl-terminal glycine residue of Urm1 was critical for the s(2) modification, indicating direct involvement of the unique ubiquitin-related system in this process. We also demonstrated that the s(2) and mcm(5) modifications in cytosolic tRNAs influence each other's efficiency. Taken together, our data indicate that the s(2) modification of cytosolic tRNAs is a more complex process that requires additional unidentified components.

Antarctic fish mitochondrial genomes lack ND6 gene.

Mitochondrial gene content shows extensive variation among eukaryotes, but is remarkably compact and static in bilateral animals. Mitochondrial genomes of bilaterians typically contain two rRNA, 22 tRNA, and 13 protein-coding genes. In this study, we report that the mitochondrial genomes of Antarctic fishes of the suborder Notothenioidei (Perciformes) lack two adjacent genes encoding NADH 6 dehydrogenase (ND6) and tRNA(Glu). Loss of the ND6 gene is reported for the first time in an animal mitochondrial genome, and is considered an extremely rare evolutionary event. Dot blot and ND6 transcript detection analyses found no evidence of mitochondrial ND6 gene copies in heteroplasmy or of a functional ND6 gene copy in the nuclear genome, respectively. Hence, we concluded that ND6 function was lost in Antarctic notothenioids, and could be compensated for by functional changes in other proteins of the mitochondrial respiratory system.

Mitochondrial encephalomyopathy due to a novel mutation in the tRNAGlu of mitochondrial DNA.

A 14-year-old boy had exercise intolerance, weakness, ataxia, and lactic acidosis. Because his muscle biopsy showed a mosaic pattern of fibers staining intensely with the succinate dehydrogenase reaction but not at all with the cytochrome c oxidase reaction, we sequenced his mitochondrial DNA and found a novel mutation (C14680A) in the gene for tRNAGlu. The mutation was present in accessible tissues from the asymptomatic mother but not from a brother with Asperger syndrome. These data expand the clinical heterogeneity of mutations in this mitochondrial gene.

Identification of a new mtDNA mutation (14724G>A) associated with mitochondrial leukoencephalopathy.

We report a novel 14724G>A mutation in the mitochondrial tRNA glutamic acid gene in a 4-year-old boy with myopathy and leukoencephalopathy. A muscle biopsy showed cytochrome c oxidase-negative ragged-red fibers and biochemical analysis of the respiratory chain enzymes in muscle homogenate revealed partial complex I and complex IV deficiencies. The mutation, which affects the dihydrouridine arm at a conserved site, was nearly homoplasmic in muscle and heteroplasmic in blood DNA of the proband, but it was absent in peripheral leukocytes from the asymptomatic mother, sister, and two maternal aunts, suggesting that it arose de novo. This report proposes to look for variants in the mitochondrial genome when dealing with otherwise undetermined leukodystrophies of childhood.

Snapshots of tRNA sulphuration via an adenylated intermediate.

Uridine at the first anticodon position (U34) of glutamate, lysine and glutamine transfer RNAs is universally modified by thiouridylase into 2-thiouridine (s2U34), which is crucial for precise translation by restricting codon-anticodon wobble during protein synthesis on the ribosome. However, it remains unclear how the enzyme incorporates reactive sulphur into the correct position of the uridine base. Here we present the crystal structures of the MnmA thiouridylase-tRNA complex in three discrete forms, which provide snapshots of the sequential chemical reactions during RNA sulphuration. On enzyme activation, an alpha-helix overhanging the active site is restructured into an idiosyncratic beta-hairpin-containing loop, which packs the flipped-out U34 deeply into the catalytic pocket and triggers the activation of the catalytic cysteine residues. The adenylated RNA intermediate is trapped. Thus, the active closed-conformation of the complex ensures accurate sulphur incorporation into the activated uridine carbon by forming a catalytic chamber to prevent solvent from accessing the catalytic site. The structures of the complex with glutamate tRNA further reveal how MnmA specifically recognizes its three different tRNA substrates. These findings provide the structural basis for a general mechanism whereby an enzyme incorporates a reactive atom at a precise position in a biological molecule.

Role of Arc1p in the modulation of yeast glutamyl-tRNA synthetase activity.

Yeast methionyl-tRNA synthetase (MetRS) and glutamyl-tRNA synthetase (GluRS) possess N-terminal extensions that bind the cofactor Arc1p in trans. The strength of GluRS-Arc1p interaction is high enough to allow copurification of the two macromolecules in a 1:1 ratio, in contrast to MetRS. Deletion analysis from the C-terminal end of the GluRS appendix combined with previous N-terminal deletions of GluRS allows restriction of the Arc1p binding site to the 110-170 amino acid region of GluRS. This region has been shown to correspond to a novel protein-protein interaction domain present in both GluRS and Arc1p but not in MetRS [Galani, K., Grosshans, H., Deinert, K., Hurt, E. C., and Simos, G. (2001) EMBO J. 20, 6889-6898]. The GluRS apoenzyme fails to show significant kinetics of tRNA aminoacylation and charges unfractionated yeast tRNA at a level 10-fold reduced compared to Arc1p-bound GluRS. The K(m) values for tRNA(Glu) measured in the ATP-PP(i) exchange were similar for the two forms of GluRS, whereas k(cat) is increased 2-fold in the presence of Arc1p. Band-shift analysis revealed a 100-fold increase in tRNA binding affinity when Arc1p is bound to GluRS. This increase requires the RNA binding properties of the full-length Arc1p since Arc1p N domain leaves the K(d) of GluRS for tRNA unchanged. Transcripts of yeast tRNA(Glu) were poor substrates for measuring tRNA aminoacylation and could not be used to clarify whether Arc1p has a specific effect on the tRNA charging reaction.

Mitochondrial DNA sequence analysis of two mouse hepatocarcinoma cell lines.

AIM: To study genetic difference of mitochondrial DNA (mtDNA) between two hepatocarcinoma cell lines (Hca-F and Hca-P) with diverse metastatic characteristics and the relationship between mtDNA changes in cancer cells and their oncogenic phenotype. METHODS: Mitochondrial DNA D-loop, tRNA(Met+Glu+Ile) and ND3 gene fragments from the hepatocarcinoma cell lines with 1 100, 1 126 and 534 bp in length respectively were analysed by PCR amplification and restriction fragment length polymorphism techniques. The D-loop 3' end sequence of the hepatocarcinoma cell lines was determined by sequencing. RESULTS: No amplification fragment length polymorphism and restriction fragment length polymorphism were observed in tRNA(Met+Glu+Ile), ND3 and D-loop of mitochondrial DNA of the hepatocarcinoma cells. Sequence differences between Hca-F and Hca-P were found in mtDNA D-loop. CONCLUSION: Deletion mutations of mitochondrial DNA restriction fragment may not play a significant role in carcinogenesis. Genetic difference of mtDNA D-loop between Hca-F and Hca-P, which may reflect the environmental and genetic influences during tumor progression, could be linked to their tumorigenic phenotypes.

[mtDNA mutations in mouse tumors].

OBJECTIVE: To investigate variations of mtDNA in mouse tumors and to explore the relationship between mtDNA mutations and murine carcinogenesis. METHODS: Variations of D-loop, ND3 and tRNAIle + Glu + Met gene fragments of mtDNA from six mouse tumor cell lines were analyzed by PCR-RFLP and PCR-SSCP techniques. RESULTS: ND3 and tRNAIle + Glu + Met gene fragments of mtDNA from the tumors showed no variations at 27 endonuclease sites. The D-loop of mtDNA from Hca-F demonstrated an additional endonuclease site of Hinf I in contrast to the inbred mouse. Upon PCR-SSCP analysis, the D-loop of mtDNA was found to possess mutations in 4 of 6 tumors. CONCLUSION: D-loop appears to be the hot spot for tumor mtDNA mutations, which may contribute to the carcinogenesis of murine tumors.

An archaebacteria-derived glutamyl-tRNA synthetase and tRNA pair for unnatural amino acid mutagenesis of proteins in Escherichia coli.

The addition of novel amino acids to the genetic code of Escherichia coli involves the generation of an aminoacyl-tRNA synthetase and tRNA pair that is 'orthogonal', meaning that it functions independently of the synthetases and tRNAs endogenous to E.coli. The amino acid specificity of the orthogonal synthetase is then modified to charge the corresponding orthogonal tRNA with an unnatural amino acid that is subsequently incorporated into a polypeptide in response to a nonsense or missense codon. Here we report the development of an orthogonal glutamic acid synthetase and tRNA pair. The tRNA is derived from the consensus sequence obtained from a multiple sequence alignment of archaeal tRNA(Glu) sequences. The glutamyl-tRNA synthetase is from the achaebacterium Pyrococcus horikoshii. The new orthogonal pair suppresses amber nonsense codons with an efficiency roughly comparable to that of the orthogonal tyrosine pair derived from Methanococcus jannaschii, which has been used to selectively incorporate a variety of unnatural amino acids into proteins in E.coli. Development of the glutamic acid orthogonal pair increases the potential diversity of unnatural amino acid structures that may be incorporated into proteins in E.coli.

Characterization of Trp(+) reversions in Escherichia coli strain WP2uvrA.

The Escherichia coli strain WP2uvrA is widely used in general mutagenicity screening tests because of its high sensitivity to many kinds of mutagens and it serves as a supplement to the standard Salmonella typhimurium tester strains. In contrast to Salmonella His(+) revertants, E.coli Trp(+) revertants have not been characterized at the molecular level. In this study we found that in the trpE65 allele of WP2uvrA the triplet that codes for the fourth amino acid from the N-terminus of anthranilate synthetase was an ochre stop codon (TAA) instead of a glutamine codon (CAA). In spontaneous Trp(+) revertants the ochre codon had been changed to glutamine (CAA), lysine (AAA), glutamic acid (GAA), leucine (TTA), serine (TCA) or tyrosine (TAC, TAT). Since tryptophan prototrophy could also be restored by ochre suppressor mutations at the anticodon sites in the genes for tRNA(Glu) (glnU), tRNA(Lys) (lysT) and tRNA(Tyr) (tyrT, tyrU), the Trp(+) reversion system with E.coli WP2uvrA detected five types of base substitutions, A.T-->T.A, A.T-->C.G, A.T-->G.C, G.C-->A.T and G.C-->T.A. About 30-50% of Trp(+) revertants induced by N-ethyl-N'-nitro-N-nitrosoguanidine, captan and angelicin plus UVA irradiation were attributable to reversion at the trpE65 ochre locus; the others were attributable to suppressor mutations. In contrast, almost all revertants induced by N-methyl-N'-nitro-N-nitrosoguanidine, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone and furylfuramide were caused by suppressor mutations. Thus, the high mutagen sensitivity of WP2uvrA is due to several target sites consisting of A.T base pairs (trpE65, lysT) and G.C base pairs (glnU, tyrT, tyrU).

Effect of modified nucleotides on Escherichia coli tRNAGlu structure and on its aminoacylation by glutamyl-tRNA synthetase. Predominant and distinct roles of the mnm5 and s2 modifications of U34.

Overproducing Escherichia coli tRNAGlu in its homologous host results in the presence of several distinctly modified forms of this molecule that we name modivariants. The predominant tRNAGlu modivariant in wild-type E. coli contains five modified nucleosides: Psi13, mnm5s2U34, m2A37, T54 and Psi55. Four other overproduced modivariants differ from it by, respectively, either the presence of an additional Psi, or the presence of s2U34, or the lack of A37 methylation combined with either s2U34 or U34. Chemical probing reveals that the anticodon loop of the predominant modivariant is less reactive to the probes than that of the four others. Furthermore, the modivariant with neither mnm5s2U34 nor m2A37 has additional perturbations in the D- and T-arms and in the variable region. The lack of a 2-thio group in nucleoside 34, which is mnm5s2U in the predominant tRNAGlu modivariant, decreases by 520-fold the specificity of E. coli glutamyl-tRNA synthetase for tRNAGlu in the aminoacylation reaction, showing that this thio group is the identity element in the modified wobble nucleotide of E. coli tRNAGlu. The modified nucleosides content also influences the recognition of ATP and glutamate by this enzyme, and in this case also, the predominant modivariant is the one that allows the best specificity for these two substrates. These structural and kinetic properties of tRNAGlu modivariants indicate that the modification system of tRNAGlu optimizes the stability of tRNAGlu and its action as cofactor of the glutamyl-tRNA synthetase for the recognition of glutamate and ATP.