Mutación heterocigota, autosómica recesiva del gen RARS2 en una paciente colombiana de padres no consanguíneos / Autosomal recessive heterocygote mutation of the RARS2 gene in a colombian patient with non- consanguineous parents

Las nuevas metodologías de secuenciación masiva han permitido caracterizar e identificar variantes genéticas asociadas a diferentes patologías. En este trabajo se presenta el caso de una paciente con una mutación del gen RARS2 que codifica la enzima arginino-ARNt ligasa para la codificación de proteínas. Esta alteración genética se manifiesta en hipoplasia pontocerebelosa tipo 6, con una prevalencia de <1/1 000 0000, caracterizada por un cerebelo y un puente de menor tamaño asociados a un retraso grave en el neurodesarrollo. El análisis de caso permite un mejor conocimiento de enfermedades de origen genético, específicamente, de aquellas con patrones de herencia autosómicos recesivos de padres no consanguíneos. Su estudio sobre todo en lo relacionado con el ámbito familiar y socioeconómico, y su base genética, ayuda a una mejor calidad de vida de los pacientes y su familia.

The latest method of next-generation sequencing has allowed the characterization and identification of genetic variants associated to diverse pathologies. In this article, we present the case of female patient with a mutation of the RARS2 gene that encodes the enzyme for arginyl tRNA synthetase for coding of proteins. This genetic alteration manifests in pontocerebellar hypoplasia type 6, with a prevalence of <1/1,000,0000, characterized by a cerebellum and pons that are smaller in size and are associated with severe neurodevelopmental delay. The analysis of the case of this patient provides better knowledge of diseases of genetic origin; specifically, regarding genetic diseases of autosomal recessive patterns of inheritance from non-consanguineous parents. The impact of these studies; specially within the family, social, economic and genetic aspects helps provide a better quality of life for these patients and their family.

Evaluation of grade in a genotyped cohort of sporadic medullary thyroid carcinomas.

AIMS: Tumour grade and RET mutation status, especially the presence of high-risk exon 15 and 16 RET mutations, have been reported to be prognostic in patients with sporadic medullary thyroid carcinoma (MTC). The aims of our study were to evaluate the performance of two recently proposed grading systems and to assess the association between grade and genotype in a cohort of sporadic MTCs. METHODS AND RESULTS: We identified 44 sporadic MTCs. All available tumour slides were examined, and cases were assigned a grade on the basis of either mitotic count and tumour necrosis, or mitotic count, tumour necrosis, and Ki-67 proliferative index, as described in two recent studies. Additional clinicopathological features and outcome information were obtained from the pathology reports and electronic medical records. The presence of RET and RAS mutations was determined either with direct sequencing or with massively parallel sequencing. Both grading systems were prognostic for progression-free survival and disease-specific survival on univariate analysis. There was no correlation between grade and mutation status. Specifically, neither RET nor high-risk RET mutations were enriched in high-grade tumours, as assessed by either grading scheme. CONCLUSION: Our findings suggest that grade is not correlated with RET/RAS mutation status, indicating that grade and genotype may give independent prognostic information.

Knockdown of Arginyl-tRNA Synthetase Attenuates Ischemia-Induced Cerebral Cortex Injury in Rats After Middle Cerebral Artery Occlusion.

Some researchers have previously shown that RNAi knockdown of arginyl-tRNA synthetase (ArgRS) before or after a hypoxic injury can rescue animals from death, based on the model organism, C. elegans. However, there has been no study on the application of arginyl-tRNA synthetase knockdown in treating mammalian ischemic stroke, and its potential mechanism and effect on ischemic brain damage are still unknown. Here, we focused on the Rars gene, which encodes an arginyl-tRNA synthetase, and examined the effects of Rars knockdown in a permanent middle cerebral artery occlusion model in rats. To achieve this aim, adult male Sprague-Dawley (SD) rats were given right cerebral cortex injections of short hairpin RNA (shRNA) adenovirus (AV) particles to knock down arginyl-tRNA synthetase, and a non-targeting control (NTC) vector or phosphate-buffered solution served as the controls. After 4 days, the rats were exposed to permanent middle cerebral artery occlusion (pMCAO). Then, the right cerebral cortex level of arginyl-tRNA synthetase was examined, and the effects of the Rars knockdown were evaluated by differences in infarction volume, oxidative stress, blood-brain barrier, mitochondrial function, and glucose metabolism at 1 day and 3 days after MCAO. The injection of shRNA adenovirus particles successfully suppressed the expression of arginyl-tRNA synthetase in the cerebral cortex. We observed an improvement in oxidative stress, mitochondrial function, and glucose utilization and a reduction in brain edema compared with the non-targeting control rats with suppressed expression of arginyl-tRNA synthetase mRNA in the ipsilateral ischemic cortex of the brain. Our findings indicate that knockdown of arginyl-tRNA synthetase in the cerebral cortex exerted neuroprotective effects, which were achieved not only by the improvement of oxidative stress and glucose utilization but also by the maintenance of mitochondrial morphological integrity and the preservation of mitochondrial function. Knockdown of ArgRS administration could be a promising approach to protect ischemic stroke.

A glycolysis-related gene signature predicts prognosis of patients with esophageal adenocarcinoma.

BACKGROUND: Esophageal adenocarcinoma (EAC) is a growing problem with a rapidly rising incidence and carries a poor prognosis. We aimed to develop a glycolysis-related gene signature to predict the prognostic outcome of patients with EAC. RESULTS: Five genes (CLDN9, GFPT1, HMMR, RARS and STMN1) were correlated with prognosis of EAC patients. Patients were classified into high-risk and low-risk groups calculated by Cox regression analysis, based on the five gene signature risk score. The five-gene signature was an independent biomarker for prognosis and patients with low risk scores showed better prognosis. Nomogram incorporating the gene signature and clinical prognostic factors was effective in predicting the overall survival. CONCLUSION: An innovative identified glycolysis-related gene signature and an effective nomogram reliably predicted the prognosis of EAC patients. METHODS: The Cancer Genome Atlas database was investigated for the gene expression profile of EAC patients. Glycolytic gene sets difference between EAC and normal tissues were identified via Gene set enrichment analysis (GSEA). Univariate and multivariate Cox analysis were utilized to construct a prognostic gene signature. The signature was evaluated by receiver operating characteristic curves and Kaplan-Meier curves. A prognosis model integrating clinical parameters with the gene signature was established with nomogram.

Molecular evidence for the evolution of the eukaryotic mitochondrial arginyl-tRNA synthetase from the prokaryotic suborder Cystobacterineae.

The evolutionary origin of the family of eukaryotic aminoacyl-tRNA synthetases that are essential to all living organisms is a matter of debate. In order to shed molecular light on the ancient source of arginyl-tRNA synthetase, a total of 1347 eukaryotic arginyl-tRNA synthetase sequences were mined from databases and analyzed. Their multiple sequence alignment reveals a signature sequence that is characteristic of the nuclear-encoded enzyme, which is imported into mitochondria. Using this molecular beacon, the origins of this gene can be traced to modern prokaryotes. In this way, a previous phylogenetic analysis linking Myxococcus to the emergence of the eukaryotic mitochondrial arginyl-tRNA synthetase is supported by the unique existence of the molecular signature within the suborder Cystobacterineae that includes Myxococcus.

Newly acquired N-terminal extension targets threonyl-tRNA synthetase-like protein into the multiple tRNA synthetase complex.

A typical feature of eukaryotic aminoacyl-tRNA synthetases (aaRSs) is the evolutionary gain of domains at either the N- or C-terminus, which frequently mediating protein-protein interaction. TARSL2 (mouse Tarsl2), encoding a threonyl-tRNA synthetase-like protein (ThrRS-L), is a recently identified aaRS-duplicated gene in higher eukaryotes, with canonical functions in vitro, which exhibits a different N-terminal extension (N-extension) from TARS (encoding ThrRS). We found the first half of the N-extension of human ThrRS-L (hThrRS-L) is homologous to that of human arginyl-tRNA synthetase. Using the N-extension as a probe in a yeast two-hybrid screening, AIMP1/p43 was identified as an interactor with hThrRS-L. We showed that ThrRS-L is a novel component of the mammalian multiple tRNA synthetase complex (MSC), and is reliant on two leucine zippers in the N-extension for MSC-incorporation in humans, and mouse cell lines and muscle tissue. The N-extension was sufficient to target a foreign protein into the MSC. The results from a Tarsl2-deleted cell line showed that it does not mediate MSC integrity. The effect of phosphorylation at various sites of hThrRS-L on its MSC-targeting is also explored. In summary, we revealed that ThrRS-L is a bona fide component of the MSC, which is mediated by a newly evolved N-extension domain.

The RARS-MAD1L1 Fusion Gene Induces Cancer Stem Cell-like Properties and Therapeutic Resistance in Nasopharyngeal Carcinoma.

Purpose: Nasopharyngeal carcinoma (NPC) is the most common head and neck cancer in Southeast Asia. Because local recurrence and distant metastasis are still the main causes of NPC treatment failure, it is urgent to identify new tumor markers and therapeutic targets for advanced NPC.Experimental Design: RNA sequencing (RNA-seq) was applied to look for interchromosome translocation in NPC. PCR, FISH, and immunoprecipitation were used to examine the fusion gene expression at RNA, DNA, and protein levels in NPC biopsies. MTT assay, colony formation assay, sphere formation assay, co-immunoprecipitation, chromatin immunoprecipitation assay, and in vivo chemoresistance assay were applied to explore the function of RARS-MAD1L1 in NPC.Results: We demonstrated that RARS-MAD1L1 was present in 10.03% (35/349) primary NPC biopsies and 10.7% (9/84) in head and neck cancer (HNC) samples. RARS-MAD1L1 overexpression increased cell proliferation, colony formation, and tumorigenicity in vitro, and the silencing of endogenous RARS-MAD1L1 reduced cancer cell growth and colony formation in vitro In addition, RARS-MAD1L1 increased the side population (SP) ratio and induced chemo- and radioresistance. Furthermore RARS-MAD1L1 interacted with AIMP2, which resulted in activation of FUBP1/c-Myc pathway. The silencing of FUBP1 or the administration of a c-Myc inhibitor abrogated the cancer stem cell (CSC)-like characteristics induced by RARS-MAD1L1. The expression of c-Myc and ABCG2 was higher in RARS-MAD1L1-positive HNC samples than in negative samples.Conclusions: Our findings indicate that RARS-MAD1L1 might contribute to tumorigenesis, CSC-like properties, and therapeutic resistance, at least in part, through the FUBP1/c-Myc axis, implying that RARS-MAD1L1 might serve as an attractive target for therapeutic intervention for NPC. Clin Cancer Res; 24(3); 659-73. ©2017 AACR.

Novel mutations in WWOX, RARS2, and C10orf2 genes in consanguineous Arab families with intellectual disability.

Intellectual disability is a heterogeneous disease with many genes and mutations influencing the phenotype. Consanguineous families constitute a rich resource for the identification of rare variants causing autosomal recessive disease, due to the effects of inbreeding. Here, we examine three consanguineous Arab families, recruited in a quest to identify novel genes/mutations. All the families had multiple offspring with non-specific intellectual disability. We identified homozygosity (autozygosity) intervals in those families through SNP genotyping and whole exome sequencing, with variants filtered using Ingenuity Variant Analysis (IVA) software. The families showed heterogeneity and novel mutations in three different genes known to be associated with intellectual disability. These mutations were not found in 514 ethnically matched control chromosomes. p.G410C in WWOX, p.H530Y in RARS2, and p.I69F in C10orf2 are novel changes that affect protein function and could give new insights into the development and function of the central nervous system.

Pontocerebellar hypoplasia type 6 caused by mutations in RARS2: definition of the clinical spectrum and molecular findings in five patients.

Recessive mutations in the mitochondrial arginyl-transfer RNA synthetase (RARS2) gene have been associated with early onset encephalopathy with signs of oxidative phosphorylation defects classified as pontocerebellar hypoplasia 6. We describe clinical, neuroimaging and molecular features on five patients from three unrelated families who displayed mutations in RARS2. All patients rapidly developed a neonatal or early-infantile epileptic encephalopathy with intractable seizures. The long-term follow-up revealed a virtual absence of psychomotor development, progressive microcephaly, and feeding difficulties. Mitochondrial respiratory chain enzymes in muscle and fibroblasts were normal in two. Blood and CSF lactate was abnormally elevated in all five patients at early stages while appearing only occasionally abnormal with the progression of the disease. Cerebellar vermis hypoplasia with normal aspect of the cerebral and cerebellar hemispheres appeared within the first months of life at brain MRI. In three patients follow-up neuroimaging revealed a progressive pontocerebellar and cerebral cortical atrophy. Molecular investigations of RARS2 disclosed the c.25A>G/p.I9V and the c.1586+3A>T in family A, the c.734G>A/p.R245Q and the c.1406G>A/p.R469H in family B, and the c.721T>A/p.W241R and c.35A>G/p.Q12R in family C. Functional complementation studies in Saccharomyces cerevisiae showed that mutation MSR1-R531H (equivalent to human p.R469H) abolished respiration whereas the MSR1-R306Q strain (corresponding to p.R245Q) displayed a reduced growth on non-fermentable YPG medium. Although mutations functionally disrupted yeast we found a relatively well preserved arginine aminoacylation of mitochondrial tRNA. Clinical and neuroimaging findings are important clues to raise suspicion and to reach diagnostic accuracy for RARS2 mutations considering that biochemical abnormalities may be absent in muscle biopsy.

Intraphylum diversity and complex evolution of cyanobacterial aminoacyl-tRNA synthetases.

A comparative genomic analysis of 35 cyanobacterial strains has revealed that the gene complement of aminoacyl-tRNA synthetases (AARSs) and routes for aminoacyl-tRNA synthesis may differ among the species of this phylum. Several genes encoding AARS paralogues were identified in some genomes. In-depth phylogenetic analysis was done for each of these proteins to gain insight into their evolutionary history. GluRS, HisRS, ArgRS, ThrRS, CysRS, and Glu-Q-RS showed evidence of a complex evolutionary course as indicated by a number of inconsistencies with our reference tree for cyanobacterial phylogeny. In addition to sequence data, support for evolutionary hypotheses involving horizontal gene transfer or gene duplication events was obtained from other observations including biased sequence conservation, the presence of indels (insertions or deletions), or vestigial traces of ancestral redundant genes. We present evidences for a novel protein domain with two putative transmembrane helices recruited independently by distinct AARS in particular cyanobacteria.

Proteasomes and RARS modulate AIMP1/EMAP II secretion in human cancer cell lines.

The aminoacyl t-RNA synthetase interacting multifunctional protein (AIMP1) is the precursor of the multifunctional inflammatory cytokine endothelial monocyte-activating polypeptide II (EMAP II). We previously demonstrated that AIMP1 secretion by pituitary adenomas is inversely correlated with tumor diameter and with RARS expression, suggesting that a high amount of RARS associated with AIMP1 might prevent the secretion of the latter cytokine. In this study, we investigated the role of RARS in modulating the secretion of AIMP1 in HeLa and MCF7 cell lines and investigated the possible role of the multicatalytic protease in the cleavage of AIMP1 to generate EMAP II. Our data show that RARS over-expression impairs AIMP1 secretion by both HeLa and MCF7 cells. Moreover, proteasome inhibition impairs AIMP1 cleavage to produce EMAP II. These data indicate that RARS over-expression associates with a reduced AIMP1 secretion and that the multicatalytic protease is involved in the generation of the mature cytokine, EMAP II.

An efficient genetic screen in Drosophila to identify nuclear-encoded genes with mitochondrial function.

We conducted a screen for glossy-eye flies that fail to incorporate BrdU in the third larval instar eye disc but exhibit normal neuronal differentiation and isolated 23 complementation groups of mutants. These same phenotypes were previously seen in mutants for cytochrome c oxidase subunit Va. We have molecularly characterized six complementation groups and, surprisingly, each encodes a mitochondrial protein. Therefore, we believe our screen to be an efficient method for identifying genes with mitochondrial function.

Two forms of human cytoplasmic arginyl-tRNA synthetase produced from two translation initiations by a single mRNA.

Human cytoplasmic arginyl-tRNA synthetase (ArgRS) is a component of a macromolecular complex consisting of at least nine tRNA synthetases and three auxiliary proteins. In mammalian cells, ArgRS is present as a free protein as well as a component of the complex. Via an alignment of ArgRSs from different vertebrates, the genes encoding full-length human cytoplasmic ArgRS and an N-terminal 72-amino acid deletion mutant (hcArgRS and DeltaNhcArgRS, respectively) were subcloned and expressed in Escherichia coli. The two ArgRS products were expressed as a soluble protein in E. coli. The level of production of DeltaNhcArgRS in E. coli and its specific activity were higher than those for hcArgRS. By Western blot analysis, using an antibody against the purified DeltaNhcArgRS, the two forms of ArgRS were detected in three human cell types. The 5'-end cDNA sequence, as confirmed by 5'RACE (5'-rapid amplification of cDNA ends), contained three start codons. Through mutation of the three codons, the two human cytoplasmic ArgRSs were found to be produced in different amounts, indicating that they resulted from two different translation initiation events. Here we show evidence that two forms of human cytoplasmic ArgRS were produced from two translational initiations by a single mRNA.

miR-15a and miR-16-1 down-regulation in pituitary adenomas.

Micro RNAs (miRs) are small noncoding RNAs, functioning as antisense regulators of other RNAs. miR-15a and miR-16-1 genes are located at chromosome 13q14, a region which is frequently deleted in pituitary tumors. An inverse correlation has been shown in B cell chronic lymphocytic leukemia (B-CLL) between miR-15a and miR-16-1 expression and the expression levels of arginyl-tRNA synthetase (RARS), an enzyme which associates with the cofactor p43 in the aminoacyl-tRNA synthetase complex. When secreted, p43 regulates local inflammatory response and macrophage chemotaxis, and seems to have anti-neoplastic properties in mice. We explored miR-15a and miR-16-1 expression in 10 GH-secreting and in 10 PRL-secreting pituitary macroadenomas by Northern blot, and investigated the possible correlation with in vivo and in vitro characteristics. We found that miR-15a and miR-16-1 are expressed at lower levels in pituitary adenomas as compared to normal pituitary tissue. Moreover, their expression inversely correlates with tumor diameter and with RARS expression (P < 0.05), but directly correlates with p43 secretion (P < 0.02). Therefore, miR15 and miR16 down-regulation in pituitary adenomas correlates with a greater tumor diameter and a lower p43 secretion, suggesting that these genes may, at least in part, influence tumor growth.

The tRNA-interacting factor p43 associates with mammalian arginyl-tRNA synthetase but does not modify its tRNA aminoacylation properties.

Arginyl-tRNA synthetase (ArgRS) is one of the nine synthetase components of a multienzyme complex containing three auxiliary proteins as well. We previously established that the N-terminal moiety of the auxiliary protein p43 associates with the N-terminal, eukaryotic-specific polypeptide extension of ArgRS. Because p43 is homologous to Arc1p, a yeast general RNA-binding protein that associates with MetRS and GluRS and plays the role of tRNA-binding cofactor in the aminoacylation reaction, we analyzed the functional significance of p43-ArgRS association. We had previously showed that full-length ArgRS, corresponding to the ArgRS species associated within the multisynthetase complex, and ArgRS with a deletion of 73 N-terminal amino acid residues, corresponding to a free species of ArgRS, both produced in yeast, have similar catalytic parameters (Lazard, M., Kerjan, P., Agou, F., and Mirande, M. (2000) J. Mol. Biol. 302, 991-1004). However, a recent study had suggested that association of p43 to ArgRS reduces the apparent K(M) of ArgRS to tRNA (Park, S. G., Jung, K. H., Lee, J. S., Jo, Y. J., Motegi, H., Kim, S., and Shiba, K. (1999) J. Biol. Chem. 274, 16673-16676). In this study, we analyzed in detail, by gel retardation assays and enzyme kinetics, the putative role of p43 as a tRNA-binding cofactor of ArgRS. The association of p43 with ArgRS neither strengthened tRNA-binding nor changed kinetic parameters in the amino acid activation or in the tRNA aminoacylation reaction. Furthermore, selective removal of the C-terminal RNA-binding domain of p43 from the multisynthetase complex did not affect kinetic parameters for ArgRS. Therefore, p43 has a dual function. It promotes association of ArgRS to the complex via its N-terminal domain, but its C-terminal RNA-binding domain may act as a tRNA-interacting factor for an as yet unidentified component of the complex.

Arginyl-tRNA synthetase with signature sequence KMSK from Bacillus stearothermophilus.

ArgRS (arginyl-tRNA synthetase) belongs to the class I aaRSs (aminoacyl-tRNA synthetases), though the majority of ArgRS species lack the canonical KMSK sequence characteristic of class I aaRSs. A DNA fragment of the ArgRS gene from Bacillus stearothermophilus was amplified using primers designed according to the conserved regions of known ArgRSs. Through analysis of the amplified DNA sequence and known tRNA(Arg)s with a published genomic sequence of B. stearothermophilus, the gene encoding ArgRS ( argS ') was amplified by PCR and the gene encoding tRNA(Arg) (ACG) was synthesized. ArgRS contained 557 amino acid residues including the canonical KMKS sequence. Recombinant ArgRS and tRNA(Arg) (ACG) were expressed in Escherichia coli. ArgRS purified by nickel-affinity chromatography had no ATPase activity. The kinetics of ArgRS and cross-recognition between ArgRSs and tRNA(Arg)s from B. stearothermophilus and E. coli were studied. The activities of B. stearothermophilus ArgRS mutated at Lys(382) and Lys(385) of the KMSK sequence and at Gly(136) upstream of the HIGH loop were determined. From the mutation results, we concluded that there was mutual compensation of Lys(385) and Gly(136) for the amino acid-activation activity of B. stearothermophilus ArgRS.

The tRNA-dependent activation of arginine by arginyl-tRNA synthetase requires inter-domain communication.

The tRNA-dependent amino acid activation catalyzed by mammalian arginyl-tRNA synthetase has been characterized. A conditional lethal mutant of Chinese hamster ovary cells that exhibits reduced arginyl-tRNA synthetase activity (Arg-1), and two of its derived revertants (Arg-1R4 and Arg-1R5) were analyzed at the structural and functional levels. A single nucleotide change, resulting in a Cys to Tyr substitution at position 599 of arginyl-tRNA synthetase, is responsible for the defective phenotype of the thermosensitive and arginine hyper-auxotroph Arg-1 cell line. The two revertants have a single additional mutation resulting in a Met222 to Ile change for Arg-1R4 or a Tyr506 to Ser change for Arg-1R5. The corresponding mutant enzymes were expressed in yeast and purified. The Cys599 to Tyr mutation affects both the thermal stability of arginyl-tRNA synthetase and the kinetic parameters for arginine in the ATP-PP(i) exchange and tRNA aminoacylation reactions. This mutation is located underneath the floor of the Rossmann fold catalytic domain characteristic of class 1 aminoacyl-tRNA synthetases, near the end of a long helix belonging to the alpha-helix bundle C-terminal domain distinctive of class 1a synthetases. For the Met222 to Ile revertant, there is very little effect of the mutation on the interaction of arginyl-tRNA synthetase with either of its substrates. However, this mutation increases the thermal stability of arginyl-tRNA synthetase, thereby leading to reversion of the thermosensitive phenotype by increasing the steady-state level of the enzyme in vivo. In contrast, for the Arg-1R5 cell line, reversion of the phenotype is due to an increased catalytic efficiency of the C599Y/Y506S double mutant as compared to the initial C599Y enzyme. In light of the location of the mutations in the 3D structure of the enzyme modeled using the crystal structure of the closely related yeast arginyl-tRNA synthetase, the kinetic analysis of these mutants suggests that the obligatory tRNA-induced activation of the catalytic site of arginyl-tRNA synthetase involves interdomain signal transduction via the long helices that build the tRNA-binding domain of the enzyme and link the site of interaction of the anticodon domain of tRNA to the floor of the active site.

Effect of cysteine residues on the activity of arginyl-tRNA synthetase from Escherichia coli.

Arginyl-tRNA synthetase (ArgRS) from Escherichia coli (E. coli) contains four cysteine residues. In this study, the role of cysteine residues in the enzyme has been investigated by chemical modification and site-directed mutagenesis. Titration of sulfhydryl groups in ArgRS by 5, 5'-dithiobis(2-nitro benzoic acid) (DTNB) suggested that a disulfide bond was not formed in the enzyme and that, in the native condition, two DTNB-sensitive cysteine residues were located on the surface of ArgRS, while the other two were buried inside. Chemical modification of the native enzyme by iodoacetamide (IAA) affected only one DTNB-sensitive cysteine residue and resulted in 50% loss of enzyme activity, while modification by N-ethylmeimide (NEM) affected two DTNB-sensitive residues and caused a complete loss of activity. These results, when combined with substrate protection experiments, suggested that at least the two cysteine residues located on the surface of the molecule were directly involved in substrates binding and catalysis. However, changing Cys to Ala only resulted in slight loss of enzymatic activity and substrate binding, suggesting that these four cysteine residues in E. coli ArgRS were not essential to the enzymatic activity. Moreover, modifications of the mutant enzymes indicated that the two DTNB- and NEM-sensitive residues were Cys(320) and Cys(537) and the IAA-sensitive was Cys(320). Our study suggested that inactivation of E. coli ArgRS by sulfhydryl reagents is a result of steric hindrance in the enzyme.

tRNA synthetase mutants of Escherichia coli K-12 are resistant to the gyrase inhibitor novobiocin.

In previous studies we demonstrated that mutations in the genes cysB, cysE, and cls (nov) affect resistance of Escherichia coli to novobiocin (J. Rakonjac, M. Milic, and D. J. Savic, Mol. Gen. Genet. 228:307-311, 1991; R. Ivanisevic, M. Milic, D. Ajdic, J. Rakonjac, and D. J. Savic, J. Bacteriol. 177:1766-1771, 1995). In this work we expand this list with mutations in rpoN (the gene for RNA polymerase subunit sigma54) and the tRNA synthetase genes alaS, argS, ileS, and leuS. Similarly to resistance to the penicillin antibiotic mecillinam, resistance to novobiocin of tRNA synthetase mutants appears to depend upon the RelA-mediated stringent response. However, at this point the overlapping pathways of mecillinam and novobiocin resistance diverge. Under conditions of stringent response induction, either by the presence of tRNA synthetase mutations or by constitutive production of RelA protein, inactivation of the cls gene diminishes resistance to novobiocin but not to mecillinam.

Cloning and characterization of cDNA encoding a human arginyl-tRNA synthetase.

Arginyl-tRNA synthetase (ArgRS) plays a key role in protein synthesis as part of a multienzyme complex with a number of other aminoacyl-tRNA synthetase (aaRS) enzymes. We have isolated a full-length cDNA encoding ArgRS as part of a project on complementation of radiosensitivity in human cells with an Epstein-Barr Virus (EBV) vector-based human cDNA library. DNA sequence analysis identified an open reading frame of 1983 nucleotides with 87% homology to other mammalian ArgRS genes. The deduced amino acid (aa) sequence (661 aa) showed 87.7% identity to the Chinese hamster ovary (CHO) enzyme and 37.7% identity to the homologous Escherichia coli enzyme. Northern blot analysis revealed the presence of a single mRNA species of approx. 2.2 kb. The results described here demonstrate that ArgRS is highly conserved in mammalian cells and confirm the presence of a hydrophobic N-terminal region in the higher-molecular-weight complexed form of ArgRS.