Regulation of ex-translational activities is the primary function of the multi-tRNA synthetase complex.

During mRNA translation, tRNAs are charged by aminoacyl-tRNA synthetases and subsequently used by ribosomes. A multi-enzyme aminoacyl-tRNA synthetase complex (MSC) has been proposed to increase protein synthesis efficiency by passing charged tRNAs to ribosomes. An alternative function is that the MSC repurposes specific synthetases that are released from the MSC upon cues for functions independent of translation. To explore this, we generated mammalian cells in which arginyl-tRNA synthetase and/or glutaminyl-tRNA synthetase were absent from the MSC. Protein synthesis, under a variety of stress conditions, was unchanged. Most strikingly, levels of charged tRNAArg and tRNAGln remained unchanged and no ribosome pausing was observed at codons for arginine and glutamine. Thus, increasing or regulating protein synthesis efficiency is not dependent on arginyl-tRNA synthetase and glutaminyl-tRNA synthetase in the MSC. Alternatively, and consistent with previously reported ex-translational roles requiring changes in synthetase cellular localizations, our manipulations of the MSC visibly changed localization.

The molecular aetiology of tRNA synthetase depletion: induction of a GCN4 amino acid starvation response despite homeostatic maintenance of charged tRNA levels.

During protein synthesis, charged tRNAs deliver amino acids to translating ribosomes, and are then re-charged by tRNA synthetases (aaRS). In humans, mutant aaRS cause a diversity of neurological disorders, but their molecular aetiologies are incompletely characterised. To understand system responses to aaRS depletion, the yeast glutamine aaRS gene (GLN4) was transcriptionally regulated using doxycycline by tet-off control. Depletion of Gln4p inhibited growth, and induced a GCN4 amino acid starvation response, indicative of uncharged tRNA accumulation and Gcn2 kinase activation. Using a global model of translation that included aaRS recharging, Gln4p depletion was simulated, confirming slowed translation. Modelling also revealed that Gln4p depletion causes negative feedback that matches translational demand for Gln-tRNAGln to aaRS recharging capacity. This maintains normal charged tRNAGln levels despite Gln4p depletion, confirmed experimentally using tRNA Northern blotting. Model analysis resolves the paradox that Gln4p depletion triggers a GCN4 response, despite maintenance of tRNAGln charging levels, revealing that normally, the aaRS population can sequester free, uncharged tRNAs during aminoacylation. Gln4p depletion reduces this sequestration capacity, allowing uncharged tRNAGln to interact with Gcn2 kinase. The study sheds new light on mutant aaRS disease aetiologies, and explains how aaRS sequestration of uncharged tRNAs can prevent GCN4 activation under non-starvation conditions.

Boosting activity of high-fidelity CRISPR/Cas9 variants using a tRNAGln-processing system in human cells.

CRISPR/Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. High-fidelity Cas9 variants have been identified; however, they often have reduced activity, constraining their utility, which presents a major challenge for their use in research applications and therapeutics. Here we developed a tRNAGln-processing system to restore the activity of multiple high-fidelity Cas9 variants in human cells, including SpCas9-HF1, eSpCas9, and xCas9. Specifically, acting on previous observations that small guide RNAs (sgRNAs) harboring an extra A or G (A/G) in the first 5' nucleotide greatly affect the activity of high-fidelity Cas9 variants and that tRNA-sgRNA fusions improve Cas9 activity, we investigated whether a GN20 sgRNA fused to different tRNAs (G-tRNA-N20) could restore the activity of SpCas9 variants in human cells. Using flow cytometry, a T7E1 assay, deep sequencing-based DNA cleavage activity assays, and HEK-293 cells, we observed that a tRNAGln-sgRNA fusion system enhanced the activity of Cas9 variants, which could be harnessed for efficient correction of a pathogenic mutation in the retinoschisin 1 (RS1) gene, resulting in 6- to 8-fold improved Cas9 activity. We propose that the tRNA-processing system developed here specifically for human cells could facilitate high-fidelity Cas9-mediated human genome-editing applications.

Bacillus subtilis exhibits MnmC-like tRNA modification activities.

The MnmE-MnmG complex of Escherichia coli uses either ammonium or glycine as a substrate to incorporate the 5-aminomethyl or 5-carboxymethylaminomethyl group into the wobble uridine of certain tRNAs. Both modifications can be converted into a 5-methylaminomethyl group by the independent oxidoreductase and methyltransferase activities of MnmC, which respectively reside in the MnmC(o) and MnmC(m) domains of this bifunctional enzyme. MnmE and MnmG, but not MnmC, are evolutionarily conserved. Bacillus subtilis lacks genes encoding MnmC(o) and/or MnmC(m) homologs. The glycine pathway has been considered predominant in this typical gram-positive species because only the 5-carboxymethylaminomethyl group has been detected in tRNALysUUU and bulk tRNA to date. Here, we show that the 5-methylaminomethyl modification is prevalent in B. subtilis tRNAGlnUUG and tRNAGluUUC. Our data indicate that B. subtilis has evolved MnmC(o)- and MnmC(m)-like activities that reside in non MnmC homologous protein(s), which suggests that both activities provide some sort of biological advantage.

Two enzymes bound to one transfer RNA assume alternative conformations for consecutive reactions.

In most bacteria and all archaea, glutamyl-tRNA synthetase (GluRS) glutamylates both tRNA(Glu) and tRNA(Gln), and then Glu-tRNA(Gln) is selectively converted to Gln-tRNA(Gln) by a tRNA-dependent amidotransferase. The mechanisms by which the two enzymes recognize their substrate tRNA(s), and how they cooperate with each other in Gln-tRNA(Gln) synthesis, remain to be determined. Here we report the formation of the 'glutamine transamidosome' from the bacterium Thermotoga maritima, consisting of tRNA(Gln), GluRS and the heterotrimeric amidotransferase GatCAB, and its crystal structure at 3.35 A resolution. The anticodon-binding body of GluRS recognizes the common features of tRNA(Gln) and tRNA(Glu), whereas the tail body of GatCAB recognizes the outer corner of the L-shaped tRNA(Gln) in a tRNA(Gln)-specific manner. GluRS is in the productive form, as its catalytic body binds to the amino-acid-acceptor arm of tRNA(Gln). In contrast, GatCAB is in the non-productive form: the catalytic body of GatCAB contacts that of GluRS and is located near the acceptor stem of tRNA(Gln), in an appropriate site to wait for the completion of Glu-tRNA(Gln) formation by GluRS. We identified the hinges between the catalytic and anticodon-binding bodies of GluRS and between the catalytic and tail bodies of GatCAB, which allow both GluRS and GatCAB to adopt the productive and non-productive forms. The catalytic bodies of the two enzymes compete for the acceptor arm of tRNA(Gln) and therefore cannot assume their productive forms simultaneously. The transition from the present glutamylation state, with the productive GluRS and the non-productive GatCAB, to the putative amidation state, with the non-productive GluRS and the productive GatCAB, requires an intermediate state with the two enzymes in their non-productive forms, for steric reasons. The proposed mechanism explains how the transamidosome efficiently performs the two consecutive steps of Gln-tRNA(Gln) formation, with a low risk of releasing the unstable intermediate Glu-tRNA(Gln).

A DNA break inducer activates the anticodon nuclease RloC and the adaptive immunity in Acinetobacter baylyi ADP1.

Double-stranded DNA breaks (DSB) cause bacteria to augment expression of DNA repair and various stress response proteins. A puzzling exception educes the anticodon nuclease (ACNase) RloC, which resembles the DSB responder Rad50 and the antiviral, translation-disabling ACNase PrrC. While PrrC's ACNase is regulated by a DNA restriction-modification (R-M) protein and a phage anti-DNA restriction peptide, RloC has an internal ACNase switch comprising a putative DSB sensor and coupled ATPase. Further exploration of RloC's controls revealed, first, that its ACNase is stabilized by the activating DNA and hydrolysed nucleotide. Second, DSB inducers activated RloC's ACNase in heterologous contexts as well as in a natural host, even when R-M deficient. Third, the DSB-induced activation of the indigenous RloC led to partial and temporary disruption of tRNA(Glu) and tRNA(Gln). Lastly, accumulation of CRISPR-derived RNA that occurred in parallel raises the possibility that the adaptive immunity and RloC provide the genotoxicated host with complementary protection from impending infections.

New insights into the incorporation of natural suppressor tRNAs at stop codons in Saccharomyces cerevisiae.

Stop codon readthrough may be promoted by the nucleotide environment or drugs. In such cases, ribosomes incorporate a natural suppressor tRNA at the stop codon, leading to the continuation of translation in the same reading frame until the next stop codon and resulting in the expression of a protein with a new potential function. However, the identity of the natural suppressor tRNAs involved in stop codon readthrough remains unclear, precluding identification of the amino acids incorporated at the stop position. We established an in vivo reporter system for identifying the amino acids incorporated at the stop codon, by mass spectrometry in the yeast Saccharomyces cerevisiae. We found that glutamine, tyrosine and lysine were inserted at UAA and UAG codons, whereas tryptophan, cysteine and arginine were inserted at UGA codon. The 5' nucleotide context of the stop codon had no impact on the identity or proportion of amino acids incorporated by readthrough. We also found that two different glutamine tRNA(Gln) were used to insert glutamine at UAA and UAG codons. This work constitutes the first systematic analysis of the amino acids incorporated at stop codons, providing important new insights into the decoding rules used by the ribosome to read the genetic code.

The output of the tRNA modification pathways controlled by the Escherichia coli MnmEG and MnmC enzymes depends on the growth conditions and the tRNA species.

In Escherichia coli, the MnmEG complex modifies transfer RNAs (tRNAs) decoding NNA/NNG codons. MnmEG catalyzes two different modification reactions, which add an aminomethyl (nm) or carboxymethylaminomethyl (cmnm) group to position 5 of the anticodon wobble uridine using ammonium or glycine, respectively. In tRNA(cmnm5s2UUG)(Gln) and tRNA(cmnm5UmAA)(Leu), however, cmnm(5) appears as the final modification, whereas in the remaining tRNAs, the MnmEG products are converted into 5-methylaminomethyl (mnm(5)) through the two-domain, bi-functional enzyme MnmC. MnmC(o) transforms cmnm(5) into nm(5), whereas MnmC(m) converts nm(5) into mnm(5), thus producing an atypical network of modification pathways. We investigate the activities and tRNA specificity of MnmEG and the MnmC domains, the ability of tRNAs to follow the ammonium or glycine pathway and the effect of mnmC mutations on growth. We demonstrate that the two MnmC domains function independently of each other and that tRNA(cmnm5s2UUG)(Gln) and tRNA(cmnm5UmAA)(Leu), are substrates for MnmC(m), but not MnmC(o). Synthesis of mnm(5)s(2)U by MnmEG-MnmC in vivo avoids build-up of intermediates in tRNA(mnm5s2UUU)(Lys). We also show that MnmEG can modify all the tRNAs via the ammonium pathway. Strikingly, the net output of the MnmEG pathways in vivo depends on growth conditions and tRNA species. Loss of any MnmC activity has a biological cost under specific conditions.

A nondiscriminating glutamyl-tRNA synthetase in the plasmodium apicoplast: the first enzyme in an indirect aminoacylation pathway.

The malaria parasite Plasmodium falciparum and related organisms possess a relict plastid known as the apicoplast. Apicoplast protein synthesis is a validated drug target in malaria because antibiotics that inhibit translation in prokaryotes also inhibit apicoplast protein synthesis and are sometimes used for malaria prophylaxis or treatment. We identified components of an indirect aminoacylation pathway for Gln-tRNA(Gln) biosynthesis in Plasmodium that we hypothesized would be essential for apicoplast protein synthesis. Here, we report our characterization of the first enzyme in this pathway, the apicoplast glutamyl-tRNA synthetase (GluRS). We expressed the recombinant P. falciparum enzyme in Escherichia coli, showed that it is nondiscriminating because it glutamylates both apicoplast tRNA(Glu) and tRNA(Gln), determined its kinetic parameters, and demonstrated its inhibition by a known bacterial GluRS inhibitor. We also localized the Plasmodium berghei ortholog to the apicoplast in blood stage parasites but could not delete the PbGluRS gene. These data show that Gln-tRNA(Gln) biosynthesis in the Plasmodium apicoplast proceeds via an essential indirect aminoacylation pathway that is reminiscent of bacteria and plastids.

Evolutionary insights about bacterial GlxRS from whole genome analyses: is GluRS2 a chimera?

BACKGROUND: Evolutionary histories of glutamyl-tRNA synthetase (GluRS) and glutaminyl-tRNA synthetase (GlnRS) in bacteria are convoluted. After the divergence of eubacteria and eukarya, bacterial GluRS glutamylated both tRNAGln and tRNAGlu until GlnRS appeared by horizontal gene transfer (HGT) from eukaryotes or a duplicate copy of GluRS (GluRS2) that only glutamylates tRNAGln appeared. The current understanding is based on limited sequence data and not always compatible with available experimental results. In particular, the origin of GluRS2 is poorly understood. RESULTS: A large database of bacterial GluRS, GlnRS, tRNAGln and the trimeric aminoacyl-tRNA-dependent amidotransferase (gatCAB), constructed from whole genomes by functionally annotating and classifying these enzymes according to their mutual presence and absence in the genome, was analyzed. Phylogenetic analyses showed that the catalytic and the anticodon-binding domains of functional GluRS2 (as in Helicobacter pylori) were independently acquired from evolutionarily distant hosts by HGT. Non-functional GluRS2 (as in Thermotoga maritima), on the other hand, was found to contain an anticodon-binding domain appended to a gene-duplicated catalytic domain. Several genomes were found to possess both GluRS2 and GlnRS, even though they share the common function of aminoacylating tRNAGln. GlnRS was widely distributed among bacterial phyla and although phylogenetic analyses confirmed the origin of most bacterial GlnRS to be through a single HGT from eukarya, many GlnRS sequences also appeared with evolutionarily distant phyla in phylogenetic tree. A GlnRS pseudogene could be identified in Sorangium cellulosum. CONCLUSIONS: Our analysis broadens the current understanding of bacterial GlxRS evolution and highlights the idiosyncratic evolution of GluRS2. Specifically we show that: i) GluRS2 is a chimera of mismatching catalytic and anticodon-binding domains, ii) the appearance of GlnRS and GluRS2 in a single bacterial genome indicating that the evolutionary histories of the two enzymes are distinct, iii) GlnRS is more widespread in bacteria than is believed, iv) bacterial GlnRS appeared both by HGT from eukarya and intra-bacterial HGT, v) presence of GlnRS pseudogene shows that many bacteria could not retain the newly acquired eukaryal GlnRS. The functional annotation of GluRS, without recourse to experiments, performed in this work, demonstrates the inherent and unique advantages of using whole genome over isolated sequence databases.

Immunity factors for two related tRNAGln targeting killer toxins distinguish cognate and non-cognate toxic subunits.

The cytoplasmic virus-like element pWR1A from Debaryomyces robertsiae encodes a toxin (DrT) with similarities to the Pichia acaciae killer toxin PaT, which acts by importing a toxin subunit (PaOrf2) with tRNA anticodon nuclease activity into target cells. As for PaT, loss of the tRNA methyltransferase Trm9 or overexpression of tRNA(Gln) increases DrT resistance and the amount of tRNA(Gln) is reduced upon toxin exposure or upon induced intracellular expression of the toxic DrT subunit gene DrORF3, indicating DrT and PaT to share the same in vivo target. Consistent with a specific tRNase activity of DrOrf3, the protein cleaves tRNA(Gln) but not tRNA(Glu) in vitro. Heterologous cytoplasmic expression identified DrOrf5 as the DrT specific immunity factor; it confers resistance to exogenous DrT as well as to intracellular expression of DrOrf3 and prevents tRNA depletion by the latter. The PaT immunity factor PaOrf4, a homologue of DrOrf5 disables intracellular action of both toxins. However, the DrT protection level mediated by PaOrf4 is reduced compared to DrOrf5, implying a recognition mechanism for the cognate toxic subunit, leading to incomplete toxicity suppression of similar, but non-cognate toxic subunits.

Molecular characterization of a Chinese family carrying a novel C4329A mutation in mitochondrial tRNAIle and tRNAGln genes.

BACKGROUND: Hypertension is a very common cardiovascular disease influenced by multiple genetic and environmental factors. More recently, there are some studies showed that mutations in mitochondrial DNA have been involved in its pathogenesis. In this study we did further investigations on this relationship. METHODS: Epidemiological research found a Han Chinese family with probable maternally transmitted hypertension. Sequence analysis of the whole mitochondrial DNA was detected from all the family members. And evaluations of the clinical, genetic and molecular characterization were also performed. RESULTS: Matrilineal relatives within the family exhibited varying degrees of hypertension with an onset age of 48-55 years. Sequence analysis of this pedigree showed a novel homoplasmic 4329C > G mutation located at the 3' end of the tRNAIle and tRNAGln genes that was absent from 366 Chinese controls. The cytosine (C) at 4329 position was very important in the structural formation and stabilization of functional tRNAs, which was highly conserved in mitochondria of various organisms and also contributed to the high fidelity of the acceptor arm. Cells carrying this mutation were also shown to harbor mitochondrial dysfunctions. CONCLUSIONS: The C4329G point mutation in tRNAIle and tRNAGln was involved in the pathogenesis of hypertension, perhaps in association with other modifying factors.

Structural conservation of an ancient tRNA sensor in eukaryotic glutaminyl-tRNA synthetase.

In all organisms, aminoacyl tRNA synthetases covalently attach amino acids to their cognate tRNAs. Many eukaryotic tRNA synthetases have acquired appended domains, whose origin, structure and function are poorly understood. The N-terminal appended domain (NTD) of glutaminyl-tRNA synthetase (GlnRS) is intriguing since GlnRS is primarily a eukaryotic enzyme, whereas in other kingdoms Gln-tRNA(Gln) is primarily synthesized by first forming Glu-tRNA(Gln), followed by conversion to Gln-tRNA(Gln) by a tRNA-dependent amidotransferase. We report a functional and structural analysis of the NTD of Saccharomyces cerevisiae GlnRS, Gln4. Yeast mutants lacking the NTD exhibit growth defects, and Gln4 lacking the NTD has reduced complementarity for tRNA(Gln) and glutamine. The 187-amino acid Gln4 NTD, crystallized and solved at 2.3 Å resolution, consists of two subdomains, each exhibiting an extraordinary structural resemblance to adjacent tRNA specificity-determining domains in the GatB subunit of the GatCAB amidotransferase, which forms Gln-tRNA(Gln). These subdomains are connected by an apparent hinge comprised of conserved residues. Mutation of these amino acids produces Gln4 variants with reduced affinity for tRNA(Gln), consistent with a hinge-closing mechanism proposed for GatB recognition of tRNA. Our results suggest a possible origin and function of the NTD that would link the phylogenetically diverse mechanisms of Gln-tRNA(Gln) synthesis.

Fidelity of tRNA 5'-maturation: a possible basis for the functional dependence of archaeal and eukaryal RNase P on multiple protein cofactors.

RNase P, which catalyzes tRNA 5'-maturation, typically comprises a catalytic RNase P RNA (RPR) and a varying number of RNase P proteins (RPPs): 1 in bacteria, at least 4 in archaea and 9 in eukarya. The four archaeal RPPs have eukaryotic homologs and function as heterodimers (POP5•RPP30 and RPP21•RPP29). By studying the archaeal Methanocaldococcus jannaschii RPR's cis cleavage of precursor tRNA(Gln) (pre-tRNA(Gln)), which lacks certain consensus structures/sequences needed for substrate recognition, we demonstrate that RPP21•RPP29 and POP5•RPP30 can rescue the RPR's mis-cleavage tendency independently by 4-fold and together by 25-fold, suggesting that they operate by distinct mechanisms. This synergistic and preferential shift toward correct cleavage results from the ability of archaeal RPPs to selectively increase the RPR's apparent rate of correct cleavage by 11,140-fold, compared to only 480-fold for mis-cleavage. Moreover, POP5•RPP30, like the bacterial RPP, helps normalize the RPR's rates of cleavage of non-consensus and consensus pre-tRNAs. We also show that archaeal and eukaryal RNase P, compared to their bacterial relatives, exhibit higher fidelity of 5'-maturation of pre-tRNA(Gln) and some of its mutant derivatives. Our results suggest that protein-rich RNase P variants might have evolved to support flexibility in substrate recognition while catalyzing efficient, high-fidelity 5'-processing.

Trans-kingdom rescue of Gln-tRNAGln synthesis in yeast cytoplasm and mitochondria.

Aminoacylation of transfer RNA(Gln) (tRNA(Gln)) is performed by distinct mechanisms in different kingdoms and represents the most diverged route of aminoacyl-tRNA synthesis found in nature. In Saccharomyces cerevisiae, cytosolic Gln-tRNA(Gln) is generated by direct glutaminylation of tRNA(Gln) by glutaminyl-tRNA synthetase (GlnRS), whereas mitochondrial Gln-tRNA(Gln) is formed by an indirect pathway involving charging by a non-discriminating glutamyl-tRNA synthetase and the subsequent transamidation by a specific Glu-tRNA(Gln) amidotransferase. Previous studies showed that fusion of a yeast non-specific tRNA-binding cofactor, Arc1p, to Escherichia coli GlnRS enables the bacterial enzyme to substitute for its yeast homologue in vivo. We report herein that the same fusion enzyme, upon being imported into mitochondria, substituted the indirect pathway for Gln-tRNA(Gln) synthesis as well, despite significant differences in the identity determinants of E. coli and yeast cytosolic and mitochondrial tRNA(Gln) isoacceptors. Fusion of Arc1p to the bacterial enzyme significantly enhanced its aminoacylation activity towards yeast tRNA(Gln) isoacceptors in vitro. Our study provides a mechanism by which trans-kingdom rescue of distinct pathways of Gln-tRNA(Gln) synthesis can be conferred by a single enzyme.

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.

Gln-tRNAGln synthesis in a dynamic transamidosome from Helicobacter pylori, where GluRS2 hydrolyzes excess Glu-tRNAGln.

In many bacteria and archaea, an ancestral pathway is used where asparagine and glutamine are formed from their acidic precursors while covalently linked to tRNA(Asn) and tRNA(Gln), respectively. Stable complexes formed by the enzymes of these indirect tRNA aminoacylation pathways are found in several thermophilic organisms, and are called transamidosomes. We describe here a transamidosome forming Gln-tRNA(Gln) in Helicobacter pylori, an ε-proteobacterium pathogenic for humans; this transamidosome displays novel properties that may be characteristic of mesophilic organisms. This ternary complex containing the non-canonical GluRS2 specific for Glu-tRNA(Gln) formation, the tRNA-dependent amidotransferase GatCAB and tRNA(Gln) was characterized by dynamic light scattering. Moreover, we observed by interferometry a weak interaction between GluRS2 and GatCAB (K(D) = 40 ± 5 µM). The kinetics of Glu-tRNA(Gln) and Gln-tRNA(Gln) formation indicate that conformational shifts inside the transamidosome allow the tRNA(Gln) acceptor stem to interact alternately with GluRS2 and GatCAB despite their common identity elements. The integrity of this dynamic transamidosome depends on a critical concentration of tRNA(Gln), above which it dissociates into separate GatCAB/tRNA(Gln) and GluRS2/tRNA(Gln) complexes. Ester bond protection assays show that both enzymes display a good affinity for tRNA(Gln) regardless of its aminoacylation state, and support a mechanism where GluRS2 can hydrolyze excess Glu-tRNA(Gln), ensuring faithful decoding of Gln codons.

Crucial contribution of the multiple copies of the initiator tRNA genes in the fidelity of tRNA(fMet) selection on the ribosomal P-site in Escherichia coli.

The accuracy of the initiator tRNA (tRNA(fMet)) selection in the ribosomal P-site is central to the fidelity of protein synthesis. A highly conserved occurrence of three consecutive G-C base pairs in the anticodon stem of tRNA(fMet) contributes to its preferential selection in the P-site. In a genetic screen, using a plasmid borne copy of an inactive tRNA(fMet) mutant wherein the three G-C base pairs were changed, we isolated Escherichia coli strains that allow efficient initiation with the tRNA(fMet) mutant. Here, extensive characterization of two such strains revealed novel mutations in the metZWV promoter severely compromising tRNA(fMet) levels. Low cellular abundance of the chromosomally encoded tRNA(fMet) allows efficient initiation with the tRNA(fMet) mutant and an elongator tRNA(Gln), revealing that a high abundance of the cellular tRNA(fMet) is crucial for the fidelity of initiator tRNA selection on the ribosomal P-site in E. coli. We discuss possible implications of the changes in the cellular tRNA(fMet) abundance in proteome remodeling.

GCN2 kinase-mediated upregulation of ubiquitin C maintains intracellular glutamine level and tRNAGln (CUG) charging under amino acid starvation.

Each tRNA is aminoacylated (charged) with a genetic codon-specific amino acid. It remains unclear what factors are associated with tRNA charging and how tRNA charging is maintained. By using the individual tRNA acylation PCR method, we found that the charging ratio of tRNAGln (CUG) reflects cellular glutamine level. When uncharged tRNAGln (CUG) increased under amino acid starvation, the kinase GCN2, which is a key stimulator of the integrated stress response, was activated. Activation of GCN2 led to the upregulation of ubiquitin C (UBC) expression. Upregulated UBC, in turn, suppressed the further reduction in tRNAGln (CUG) charging levels. Thus, tRNA charging is sensitive to intracellular nutrient status and is an important initiator of intracellular signaling.

Assessment of tRNAThr and tRNAGln Variants and Mitochondrial Functionality in Parkinson's Disease (PD) Patients of Tamil Nadu Population.

Parkinson's disease (PD) is speculated with genetic and environmental factors. At molecular level, the mitochondrial impact is stated to be one of the causative reasons for PD. In this study, we investigated the mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and adenosine triphosphate (ATP) levels along with mitochondrial tRNA alterations among three age categories of PD. By determining the genetic and organellar functionality using molecular techniques, the ROS levels were reported to be high with decreased MMP and ATP in the late-onset age group than in other two age categories. Likewise, the tRNA significancy in tRNAThr and tRNAGln was noticed with C4335T and G15927A mutations in late-onset and early-onset PD groups respectively. Therefore, from the findings, ageing has shown a disruption in tRNA metabolism leading to critical functioning of ATP synthesis and MMP, causing oxidative stress in PD patients. These physiological outcomes show that ageing has a keen role in the divergence of mitochondrial function, thereby proving a correlation with ageing and maintenance of mitochondrial homeostasis in PD.