Short tRNA anticodon stem and mutant eRF1 allow stop codon reassignment.

Cognate tRNAs deliver specific amino acids to translating ribosomes according to the standard genetic code, and three codons with no cognate tRNAs serve as stop codons. Some protists have reassigned all stop codons as sense codons, neglecting this fundamental principle1-4. Here we analyse the in-frame stop codons in 7,259 predicted protein-coding genes of a previously undescribed trypanosomatid, Blastocrithidia nonstop. We reveal that in this species in-frame stop codons are underrepresented in genes expressed at high levels and that UAA serves as the only termination codon. Whereas new tRNAsGlu fully cognate to UAG and UAA evolved to reassign these stop codons, the UGA reassignment followed a different path through shortening the anticodon stem of tRNATrpCCA from five to four base pairs (bp). The canonical 5-bp tRNATrp recognizes UGG as dictated by the genetic code, whereas its shortened 4-bp variant incorporates tryptophan also into in-frame UGA. Mimicking this evolutionary twist by engineering both variants from B. nonstop, Trypanosoma brucei and Saccharomyces cerevisiae and expressing them in the last two species, we recorded a significantly higher readthrough for all 4-bp variants. Furthermore, a gene encoding B. nonstop release factor 1 acquired a mutation that specifically restricts UGA recognition, robustly potentiating the UGA reassignment. Virtually the same strategy has been adopted by the ciliate Condylostoma magnum. Hence, we describe a previously unknown, universal mechanism that has been exploited in unrelated eukaryotes with reassigned stop codons.

Human TRUB1 is a highly conserved pseudouridine synthase responsible for the formation of Ψ55 in mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro.

Pseudouridine (Ψ) at position 55 in tRNAs plays an important role in their structure and function. This modification is catalyzed by TruB/Pus4/Cbf5 family of pseudouridine synthases in bacteria and yeast. However, the mechanism of TRUB family underlying the formation of Ψ55 in the mammalian tRNAs is largely unknown. In this report, the CMC/reverse transcription assays demonstrated the presence of Ψ55 in the human mitochondrial tRNAAsn, tRNAGln, tRNAGlu, tRNAPro, tRNAMet, tRNALeu(UUR) and tRNASer(UCN). TRUB1 knockout (KO) cell lines generated by CRISPR/Cas9 technology exhibited the loss of Ψ55 modification in mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro but did not affect other 18 mitochondrial tRNAs. An in vitro assay revealed that recombinant TRUB1 protein can catalyze the efficient formation of Ψ55 in tRNAAsn and tRNAGln, but not in tRNAMet and tRNAArg. Notably, the overexpression of TRUB1 cDNA reversed the deficient Ψ55 modifications in these tRNAs in TRUB1KO HeLa cells. TRUB1 deficiency affected the base-pairing (18A/G-Ψ55), conformation and stability but not aminoacylation capacity of these tRNAs. Furthermore, TRUB1 deficiency impacted mitochondrial translation and biogenesis of oxidative phosphorylation system. Our findings demonstrated that human TRUB1 is a highly conserved mitochondrial pseudouridine synthase responsible for the Ψ55 modification in the mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro.

Role of the transfer ribonucleic acid (tRNA) bound magnesium ions in the charging step of aminoacylation reaction in the glutamyl tRNA synthetase and the seryl tRNA synthetase bound with cognate tRNA.

Aminoacylation reaction is the first step of protein biosynthesis. Transfer RNA (tRNA) is charged with an amino acid in this reaction and the reaction is catalyzed by aminoacyl tRNA synthetase enzyme (aaRS). In the present work, we use classical molecular dynamics simulation to show that the tRNA bound Mg2+ ions significantly influence the charging step of class I TtGluRS: Glu-AMP: tRNAGlu and class II dimeric TtSerRS: Ser-AMP: tRNASer. The CCA end of the acceptor terminal is disordered in the absence of coordinated Mg2+ ions and the CCA end can freely explore beyond the specific conformational space of the tRNA in its precharging state. A balance between the conformational disorder of the tRNA and the restriction imposed on the CCA terminal via coordination with the Mg2+ ions is needed for the placement of the CCA terminal in a precharging state organization. This result provides a molecular-level explanation of the experimental observation that the presence of Mg2+ ions is a necessary condition for a successful aminoacylation reaction.Communicated by Ramaswamy H. Sarma.

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.

Evaluación de pequeños ARN circulantes como potenciales biomarcadores en cáncer de pulmón / Evaluation of small circulating RNAs as potential biomarkers in lung cancer

El cáncer broncopulmonar es primero en mortalidad por cáncer, debido en parte al diagnóstico tardío, su agresividad inherente y la falta de marcadores específicos predictores de la respuesta a los tratamientos. Esto apoya la búsqueda de estrategias accesibles y poco invasivas para su detección precoz. Los pequeños ARN no codificantes (sRNA, del inglés small) son reguladores de la expresión génica involucrados en múltiples procesos biológicos, cuya expresión aberrante puede tener un rol central en la carcinogénesis. Estos pueden ser secretados al medio extracelular, circulando de forma estable en sangre y otros fluidos, donde son captados por células adyacentes o a distancia con funciones conservadas. Recientemente, se describieron nuevas clases de sRNA como las mitades derivadas de ARN de transferencia (tRNA-h, del inglés halves) y los Y-RNA (Y-ARN, nomenclatura universal en inglés). Su secreción al medio extracelular se ha vinculado a distintas respuestas regulatorias frente al estrés celular. El objetivo de este estudio fue caracterizar sRNA circulantes, 5'tRNA-h-Gly, 5'tRNA-h-Glu e Y4-RNA, como potenciales biomarcadores circulantes en el diagnóstico de cáncer de pulmón. Relacionar estos biomarcadores con determinadas características de la enfermedad. Los niveles de expresión relativa de cada marcador se cuantificaron mediante RT-qPCR, se construyeron curvas ROC para definir su valor como test diagnóstico. Se reclutaron 40 pacientes con cáncer de pulmón y 20 controles. Se observó una expresión tres veces mayor de 5'tRNA h-Gly circulante en pacientes con cáncer broncopulmonar en comparación con controles. Solo este fragmento se comportó como un potencial biomarcador circulante diagnóstico con significancia estadística. Este fue un mejor biomarcador en estadios avanzados de la enfermedad, lo que no pudo demostrarse para estadios precoces. Se observó una fuerte tendencia al aumento de las 5'tRNA-h-Gly en cáncer de pulmón no células pequeñas y de los 5'tRNA-h-Glu en tumores de células pequeñas. Es el primer estudio en analizar el rol de las 5'tRNA-h-Gly, 5'tRNA-Glu y Y4-RNA como biomarcadores circulantes en el diagnóstico de cáncer broncopulmonar. Este es un tema que requiere mayor desarrollo, por lo que las primeras aproximaciones pueden ayudar a comprender mejor su rol como moléculas circulantes y sentar las bases para estudios a mayor escala, venciendo las dificultades encontradas hasta el momento. La capacidad diagnóstica de los fragmentos estudiados debe ser analizada en una cohorte de validación con un mayor tamaño. Consideramos importante realizar en una siguiente etapa secuenciado masivo de sRNA en suero de muestras seleccionadas con el fin de identificar nuevos sRNA candidatos que no fueron analizados en este estudio

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.

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.

Dimerization confers increased stability to nucleases in 5' halves from glycine and glutamic acid tRNAs.

We have previously shown that 5' halves from tRNAGlyGCC and tRNAGluCUC are the most enriched small RNAs in the extracellular space of human cell lines, and especially in the non-vesicular fraction. Extracellular RNAs are believed to require protection by either encapsulation in vesicles or ribonucleoprotein complex formation. However, deproteinization of non-vesicular tRNA halves does not affect their retention in size-exclusion chromatography. Thus, we considered alternative explanations for their extracellular stability. In-silico analysis of the sequence of these tRNA-derived fragments showed that tRNAGly 5' halves can form homodimers or heterodimers with tRNAGlu 5' halves. This capacity is virtually unique to glycine tRNAs. By analyzing synthetic oligonucleotides by size exclusion chromatography, we provide evidence that dimerization is possible in vitro. tRNA halves with single point substitutions preventing dimerization are degraded faster both in controlled nuclease digestion assays and after transfection in cells, showing that dimerization can stabilize tRNA halves against the action of cellular nucleases. Finally, we give evidence supporting dimerization of endogenous tRNAGlyGCC 5' halves inside cells. Considering recent reports have shown that 5' tRNA halves from Ala and Cys can form tetramers, our results highlight RNA intermolecular structures as a new layer of complexity in the biology of tRNA-derived fragments.

Crystal structure of Arabidopsis thaliana glutamyl-tRNAGlu reductase in complex with NADPH and glutamyl-tRNAGlu reductase binding protein.

In higher plants, the tetrapyrrole biosynthesis pathway starts from the reaction catalyzed by the rate-limiting enzyme, glutamyl-tRNAGlu reductase (GTR). In Arabidopsis thaliana, GTR is controlled by post-transcriptional regulators such as GTR binding protein (GBP), which stimulates AtGTR activity. The NADPH-binding domain of AtGTR undergoes a substantial movement upon GBP binding. Here, we report the crystal structure of AtGTR-NADPH-GBP ternary complex. NADPH binding causes slight structural changes compared with the AtGTR-GBP binary complex, and possibly take a part of the space needed by the substrate glutamyl-tRNAGlu. The highly reactive sulfhydryl group of the active-site residue Cys144 shows an obvious rotation, which may facilitate the hydride transfer from NADPH to the thioester intermediate to form glutamate-1-semialdehyde. Furthermore, Lys271, Lys274, Ser275, Asn278, and Gln282 of GBP participate in the interaction between AtGTR and GBP, and the stimulating effect of GBP decreased when all of these residues were mutated to Ala. When the Cys144 of AtGTR was mutated to Ser, AtGTR activity could not be detected even in the presence of GBP.

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.

Crystallographic capture of a radical S-adenosylmethionine enzyme in the act of modifying tRNA.

RlmN is a dual-specificity RNA methylase that modifies C2 of adenosine 2503 (A2503) in 23S rRNA and C2 of adenosine 37 (A37) in several Escherichia coli transfer RNAs (tRNAs). A related methylase, Cfr, modifies C8 of A2503 via a similar mechanism, conferring resistance to multiple classes of antibiotics. Here, we report the x-ray structure of a key intermediate in the RlmN reaction, in which a Cys(118)→Ala variant of the protein is cross-linked to a tRNA(Glu)substrate through the terminal methylene carbon of a formerly methylcysteinyl residue and C2 of A37. RlmN contacts the entire length of tRNA(Glu), accessing A37 by using an induced-fit strategy that completely unfolds the tRNA anticodon stem-loop, which is likely critical for recognition of both tRNA and ribosomal RNA substrates.

A dual fluorescent reporter for the investigation of methionine mistranslation in live cells.

In mammalian cells under oxidative stress, the methionyl-tRNA synthetase (MetRS) misacylates noncognate tRNAs at frequencies as high as 10% distributed among up to 28 tRNA species. Instead of being detrimental for the cell, misincorporation of methionine residues in the proteome reduces the risk of oxidative damage to proteins, which aids the oxidative stress response. tRNA microarrays have been essential for the detection of the full pattern of misacylated tRNAs, but have limited capacity to investigate the misacylation and mistranslation mechanisms in live cells. Here we develop a dual-fluorescence reporter to specifically measure methionine misincorporation at glutamic acid codons GAA and GAG via tRNA(Glu) mismethionylation in human cells. Our method relies on mutating a specific Met codon in the active site of the fluorescent protein mCherry to a Glu codon that renders mCherry nonfluorescent when translation follows the genetic code. Mistranslation utilizing mismethionylated tRNA(Glu) restores fluorescence in proportion to the amount of misacylated tRNA(Glu). This cellular approach works well for both transient transfection and established stable HEK293 lines. It is rapid, straightforward, and well suited for high-throughput activity analysis under a wide range of physiological conditions. As a proof of concept, we apply this method to characterize the effect of human tRNA(Glu) isodecoders on mistranslation and discuss the implications of our findings.

Assessment of small RNA sorting into different extracellular fractions revealed by high-throughput sequencing of breast cell lines.

Intercellular communication can be mediated by extracellular small regulatory RNAs (sRNAs). Circulating sRNAs are being intensively studied for their promising use as minimally invasive disease biomarkers. To date, most attention is centered on exosomes and microRNAs as the vectors and the secreted species, respectively. However, this field would benefit from an increased understanding of the plethora of sRNAs secreted by different cell types in different extracellular fractions. It is still not clear if specific sRNAs are selected for secretion, or if sRNA secretion is mostly passive. We sequenced the intracellular sRNA content (19-60 nt) of breast epithelial cell lines (MCF-7 and MCF-10A) and compared it with extracellular fractions enriched in microvesicles, exosomes and ribonucleoprotein complexes. Our results are consistent with a non-selective secretion model for most microRNAs, although a few showed secretion patterns consistent with preferential secretion. On the contrary, 5' tRNA halves and 5' RNA Y4-derived fragments of 31-33 were greatly and significantly enriched in the extracellular space (even in non-mammary cell lines), where tRNA halves were detected as part of ∼45 kDa ribonucleoprotein complexes. Overall, we show that different sRNA families have characteristic secretion patterns and open the question of the role of these sRNAs in the extracellular space.

tRNAGlu increases the affinity of glutamyl-tRNA synthetase for its inhibitor glutamyl-sulfamoyl-adenosine, an analogue of the aminoacylation reaction intermediate glutamyl-AMP: mechanistic and evolutionary implications.

For tRNA-dependent protein biosynthesis, amino acids are first activated by aminoacyl-tRNA synthetases (aaRSs) yielding the reaction intermediates aminoacyl-AMP (aa-AMP). Stable analogues of aa-AMP, such as aminoacyl-sulfamoyl-adenosines, inhibit their cognate aaRSs. Glutamyl-sulfamoyl-adenosine (Glu-AMS) is the best known inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS). Thermodynamic parameters of the interactions between Glu-AMS and E. coli GluRS were measured in the presence and in the absence of tRNA by isothermal titration microcalorimetry. A significant entropic contribution for the interactions between Glu-AMS and GluRS in the absence of tRNA or in the presence of the cognate tRNAGlu or of the non-cognate tRNAPhe is indicated by the negative values of -TΔSb, and by the negative value of ΔCp. On the other hand, the large negative enthalpy is the dominant contribution to ΔGb in the absence of tRNA. The affinity of GluRS for Glu-AMS is not altered in the presence of the non-cognate tRNAPhe, but the dissociation constant Kd is decreased 50-fold in the presence of tRNAGlu; this result is consistent with molecular dynamics results indicating the presence of an H-bond between Glu-AMS and the 3'-OH oxygen of the 3'-terminal ribose of tRNAGlu in the Glu-AMS•GluRS•tRNAGlu complex. Glu-AMS being a very close structural analogue of Glu-AMP, its weak binding to free GluRS suggests that the unstable Glu-AMP reaction intermediate binds weakly to GluRS; these results could explain why all the known GluRSs evolved to activate glutamate only in the presence of tRNAGlu, the coupling of glutamate activation to its transfer to tRNA preventing unproductive cleavage of ATP.

Structure and mechanism of the tRNA-dependent lantibiotic dehydratase NisB.

Lantibiotics are a class of peptide antibiotics that contain one or more thioether bonds. The lantibiotic nisin is an antimicrobial peptide that is widely used as a food preservative to combat food-borne pathogens. Nisin contains dehydroalanine and dehydrobutyrine residues that are formed by the dehydration of Ser/Thr by the lantibiotic dehydratase NisB (ref. 2). Recent biochemical studies revealed that NisB glutamylates Ser/Thr side chains as part of the dehydration process. However, the molecular mechanism by which NisB uses glutamate to catalyse dehydration remains unresolved. Here we show that this process involves glutamyl-tRNA(Glu) to activate Ser/Thr residues. In addition, the 2.9-Å crystal structure of NisB in complex with its substrate peptide NisA reveals the presence of two separate domains that catalyse the Ser/Thr glutamylation and glutamate elimination steps. The co-crystal structure also provides insights into substrate recognition by lantibiotic dehydratases. Our findings demonstrate an unexpected role for aminoacyl-tRNA in the formation of dehydroamino acids in lantibiotics, and serve as a basis for the functional characterization of the many lantibiotic-like dehydratases involved in the biosynthesis of other classes of natural products.

tRNA thiolation links translation to stress responses in Saccharomyces cerevisiae.

Although tRNA modifications have been well catalogued, the precise functions of many modifications and their roles in mediating gene expression are still being elucidated. Whereas tRNA modifications were long assumed to be constitutive, it is now apparent that the modification status of tRNAs changes in response to different environmental conditions. The URM1 pathway is required for thiolation of the cytoplasmic tRNAs tGlu(UUC), tGln(UUG), and tLys(UUU) in Saccharomyces cerevisiae. We demonstrate that URM1 pathway mutants have impaired translation, which results in increased basal activation of the Hsf1-mediated heat shock response; we also find that tRNA thiolation levels in wild-type cells decrease when cells are grown at elevated temperature. We show that defects in tRNA thiolation can be conditionally advantageous, conferring resistance to endoplasmic reticulum stress. URM1 pathway proteins are unstable and hence are more sensitive to changes in the translational capacity of cells, which is decreased in cells experiencing stresses. We propose a model in which a stress-induced decrease in translation results in decreased levels of URM1 pathway components, which results in decreased tRNA thiolation levels, which further serves to decrease translation. This mechanism ensures that tRNA thiolation and translation are tightly coupled and coregulated according to need.

Effect of hydrogen peroxide on the biosynthesis of heme and proteins: potential implications for the partitioning of Glu-tRNA(Glu) between these pathways.

Glutamyl-tRNA (Glu-tRNA(Glu)) is the common substrate for both protein translation and heme biosynthesis via the C5 pathway. Under normal conditions, an adequate supply of this aminoacyl-tRNA is available to both pathways. However, under certain circumstances, Glu-tRNA(Glu) can become scarce, resulting in competition between the two pathways for this aminoacyl-tRNA. In Acidithiobacillus ferrooxidans, glutamyl-tRNA synthetase 1 (GluRS1) is the main enzyme that synthesizes Glu-tRNA(Glu). Previous studies have shown that GluRS1 is inactivated in vitro by hydrogen peroxide (H2O2). This raises the question as to whether H2O2 negatively affects in vivo GluRS1 activity in A. ferrooxidans and whether Glu-tRNA(Glu) distribution between the heme and protein biosynthesis processes may be affected by these conditions. To address this issue, we measured GluRS1 activity. We determined that GluRS1 is inactivated when cells are exposed to H2O2, with a concomitant reduction in intracellular heme level. The effects of H2O2 on the activity of purified glutamyl-tRNA reductase (GluTR), the key enzyme for heme biosynthesis, and on the elongation factor Tu (EF-Tu) were also measured. While exposing purified GluTR, the first enzyme of heme biosynthesis, to H2O2 resulted in its inactivation, the binding of glutamyl-tRNA to EF-Tu was not affected. Taken together, these data suggest that in A. ferrooxidans, the flow of glutamyl-tRNA is diverted from heme biosynthesis towards protein synthesis under oxidative stress conditions.

The Dnmt2 RNA methyltransferase homolog of Geobacter sulfurreducens specifically methylates tRNA-Glu.

Dnmt2 enzymes are conserved in eukaryotes, where they methylate C38 of tRNA-Asp with high activity. Here, the activity of one of the very few prokaryotic Dnmt2 homologs from Geobacter species (GsDnmt2) was investigated. GsDnmt2 was observed to methylate tRNA-Asp from flies and mice. Unexpectedly, it had only a weak activity toward its matching Geobacter tRNA-Asp, but methylated Geobacter tRNA-Glu with good activity. In agreement with this result, we show that tRNA-Glu is methylated in Geobacter while the methylation is absent in tRNA-Asp. The activities of Dnmt2 enzymes from Homo sapiens, Drosophila melanogaster, Schizosaccharomyces pombe and Dictyostelium discoideum for methylation of the Geobacter tRNA-Asp and tRNA-Glu were determined showing that all these Dnmt2s preferentially methylate tRNA-Asp. Hence, the GsDnmt2 enzyme has a swapped transfer ribonucleic acid (tRNA) specificity. By comparing the different tRNAs, a characteristic sequence pattern was identified in the variable loop of all preferred tRNA substrates. An exchange of two nucleotides in the variable loop of murine tRNA-Asp converted it to the corresponding variable loop of tRNA-Glu and led to a strong reduction of GsDnmt2 activity. Interestingly, the same loss of activity was observed with human DNMT2, indicating that the variable loop functions as a specificity determinant in tRNA recognition of Dnmt2 enzymes.

In Aspergillus nidulans the suppressors suaA and suaC code for release factors eRF1 and eRF3 and suaD codes for a glutamine tRNA.

In Aspergillus nidulans, after extensive mutagenesis, a collection of mutants was obtained and four suppressor loci were identified genetically that could suppress mutations in putative chain termination mutations in different genes. Suppressor mutations in suaB and suaD have a similar restricted spectrum of suppression and suaB111 was previously shown to be an alteration in the anticodon of a gln tRNA. We have shown that like suaB, a suaD suppressor has a mutation in the anticodon of another gln tRNA allowing suppression of UAG mutations. Mutations in suaA and suaC had a broad spectrum of suppression. Four suaA mutations result in alterations in the coding region of the eukaryotic release factor, eRF1, and another suaA mutation has a mutation in the upstream region of eRF1 that prevents splicing of the first intron within the 5'UTR. Epitope tagging of eRF1 in this mutant results in 20% of the level of eRF1 compared to the wild-type. Two mutations in suaC result in alterations in the eukaryotic release factor, eRF3. This is the first description in Aspergillus nidulans of an alteration in eRF3 leading to suppression of chain termination mutations.