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Heterozygous pathogenic variants in the histidyl-tRNA synthetase (HARS) gene are associated with Charcot-Marie-Tooth (CMT) type 2W disease, classified as an axonal peripheral neuropathy. To date, at least 60 variants causing CMT symptoms have been identified in seven different aminoacyl-tRNA synthetases, with eight being found in the catalytic domain of HARS. The genetic data clearly show a causative role of aminoacyl-tRNA synthetases in CMT; however, the cellular mechanisms leading to pathology can vary widely and are unknown in the case of most identified variants. Here we describe a novel HARS variant, c.412T>C; p.Y138H, identified through a CMT gene panel in a patient with peripheral neuropathy. To determine the effect of p.Y138H we employed a humanized HARS yeast model and recombinant protein biochemistry, which identified a deficiency in protein dimerization and a growth defect which shows mild but significant improvement with histidine supplementation. This raises the potential for a clinical trial of histidine., (© 2024 The Author(s). IUBMB Life published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

Protein kinase Gcn2 attenuates protein synthesis in response to amino acid starvation while stimulating translation of a transcriptional activator of amino acid biosynthesis. Gcn2 activation requires a domain related to histidyl-tRNA synthetase (HisRS), the enzyme that aminoacylates tRNA His . While evidence suggests that deacylated tRNA binds the HisRS domain for kinase activation, ribosomal P-stalk proteins have been implicated as alternative activating ligands on stalled ribosomes. We report crystal structures of the HisRS domain of Chaetomium thermophilum Gcn2 that reveal structural mimicry of both catalytic (CD) and anticodon-binding (ABD) domains, which in authentic HisRS bind the acceptor stem and anticodon loop of tRNA His . Elements for forming histidyl adenylate and aminoacylation are lacking, suggesting that Gcn2 HisRS was repurposed for kinase activation, consistent with mutations in the CD that dysregulate yeast Gcn2 function. Substituting conserved ABD residues well positioned to contact the anticodon loop or that form a conserved ABD-CD interface impairs Gcn2 function in starved cells. Mimicry in Gcn2 HisRS of two highly conserved structural domains for binding both ends of tRNA-each crucial for Gcn2 function-supports that deacylated tRNAs activate Gcn2 and exemplifies how a metabolic enzyme is repurposed to host new local structures and sequences that confer a novel regulatory function., Competing Interests: Competing interests statement:The authors declare no competing interest.

Histidyl-tRNA synthetase (HARS) ligates histidine to its cognate transfer RNA (tRNA His ). Mutations in HARS cause the human genetic disorders Usher syndrome type 3B (USH3B) and Charcot-Marie-Tooth syndrome type 2W (CMT2W). Treatment for these diseases remains symptomatic, and no disease specific treatments are currently available. Mutations in HARS can lead to destabilization of the enzyme, reduced aminoacylation, and decreased histidine incorporation into the proteome. Other mutations lead to a toxic gain-of-function and mistranslation of non-cognate amino acids in response to histidine codons, which can be rescued by histidine supplementation in vitro. We discuss recent advances in characterizing HARS mutations and potential applications of amino acid and tRNA therapy for future gene and allele specific therapy.

Charcot-Marie-Tooth disease (CMT) encompasses a set of genetically and clinically heterogeneous neuropathies characterized by length-dependent dysfunction of the peripheral nervous system. Mutations in over 80 diverse genes are associated with CMT, and aminoacyl-tRNA synthetases (ARS) constitute a large gene family implicated in the disease. Despite considerable efforts to elucidate the mechanistic link between ARS mutations and the CMT phenotype, the molecular basis of the pathology is unknown. In this work, we investigated the impact of three CMT-associated substitutions (V155G, Y330C, and R137Q) in the cytoplasmic histidyl-tRNA synthetase (HARS1) on neurite outgrowth and peripheral nervous system development. The model systems for this work included a nerve growth factor-stimulated neurite outgrowth model in rat pheochromocytoma cells (PC12), and a zebrafish line with GFP/red fluorescent protein reporters of sensory and motor neuron development. The expression of CMT-HARS1 mutations led to attenuation of protein synthesis and increased phosphorylation of eIF2α in PC12 cells and was accompanied by impaired neurite and axon outgrowth in both models. Notably, these effects were phenocopied by histidinol, a HARS1 inhibitor, and cycloheximide, a protein synthesis inhibitor. The mutant proteins also formed heterodimers with wild-type HARS1, raising the possibility that CMT-HARS1 mutations cause disease through a dominant-negative mechanism. Overall, these findings support the hypothesis that CMT-HARS1 alleles exert their toxic effect in a neuronal context, and lead to dysregulated protein synthesis. These studies demonstrate the value of zebrafish as a model for studying mutant alleles associated with CMT, and for characterizing the processes that lead to peripheral nervous system dysfunction., (© 2020 Federation of European Biochemical Societies.)

Reprogramming of the genetic code system is limited by the difficulty in creating new tRNA structures. Here, I developed translationally active tRNA variants tagged with a small hairpin RNA aptamer, using Escherichia coli reporter assay systems. As the tRNA chassis for engineering, I employed amber suppressor variants of allo-tRNAs having the 9/3 composition of the 12-base pair amino-acid acceptor branch as well as a long variable arm (V-arm). Although their V-arm is a strong binding site for seryl-tRNA synthetase (SerRS), insertion of a bulge nucleotide in the V-arm stem region prevented allo-tRNA molecules from being charged by SerRS with serine. The SerRS-rejecting allo-tRNA chassis were engineered to have another amino-acid identity of either alanine, tyrosine, or histidine. The tip of the V-arms was replaced with diverse hairpin RNA aptamers, which were recognized by their cognate proteins expressed in E. coli . A high-affinity interaction led to the sequestration of allo-tRNA molecules, while a moderate-affinity aptamer moiety recruited histidyl-tRNA synthetase variants fused with the cognate protein domain. The new design principle for tRNA-aptamer fusions will enhance radical and dynamic manipulation of the genetic code.

Aminoacyl-tRNA synthetases (aaRSs) play essential roles in protein translation. In addition, numerous aaRSs (mostly in vertebrates) have also been discovered to possess a range of non-canonical functions. Very few studies have been conducted to elucidate or characterize non-canonical functions of plant aaRSs. A genome-wide search for aaRS genes in Arabidopsis thaliana revealed a total of 59 aaRS genes. Among them, asparaginyl-tRNA synthetase (AsnRS) was found to possess a WHEP domain inserted into the catalytic domain in a plant-specific manner. This insertion was observed only in the cytosolic isoform. In addition, a long stretch of sequence that exhibited weak homology with histidine ammonia lyase (HAL) was found at the N-terminus of histidyl-tRNA synthetase (HisRS). This HAL-like domain has only been seen in plant HisRS, and only in cytosolic isoforms. Additionally, a number of genes lacking minor or major portions of the full-length aaRS sequence were found. These genes encode 14 aaRS fragments that lack key active site sequences and are likely catalytically null. These identified genes that encode plant-specific additional domains or aaRS fragment sequences are candidates for aaRSs possessing non-canonical functions.

Mutations in histidyl-tRNA synthetase (HARS1), an enzyme that charges transfer RNA with the amino acid histidine in the cytoplasm, have only been associated to date with autosomal recessive Usher syndrome type III and autosomal dominant Charcot-Marie-Tooth disease type 2W. Using massive parallel sequencing, we identified bi-allelic HARS1 variants in a child (c.616G>T, p.Asp206Tyr and c.730delG, p.Val244Cysfs*6) and in two sisters (c.1393A>C, p.Ile465Leu and c.910_912dupTTG, p.Leu305dup), all characterized by a multisystem ataxic syndrome. All mutations are rare, segregate with the disease, and are predicted to have a significant effect on protein function. Functional studies helped to substantiate their disease-related roles. Indeed, yeast complementation assays showing that one out of two mutations in each patient is loss-of-function, and the reduction of messenger RNA and protein levels and enzymatic activity in patient's skin-derived fibroblasts, together support the pathogenicity of the identified HARS1 variants in the patient phenotypes. Thus, our efforts expand the allelic and clinical spectrum of HARS1-related disease., (© 2020 Wiley Periodicals, Inc.)

HARS2 encodes mitochondrial histidyl-tRNA synthetase (HARS2), which links histidine to its cognate tRNA in the mitochondrial matrix. Biallelic variants in HARS2 are associated with Perrault syndrome, a rare recessive condition characterized by sensorineural hearing loss in both sexes and primary ovarian insufficiency in 46,XX females. Some individuals with Perrault syndrome have a broader phenotypic spectrum with neurological features, including ataxia and peripheral neuropathy. Here, we report a recurrent variant in HARS2 in association with sensorineural hearing loss. In affected individuals from three unrelated families, the variant HARS2 c.1439G>A p.(Arg480His) is present as a heterozygous variant in trans to a putative pathogenic variant. The low prevalence of the allele HARS2 c.1439G>A p.(Arg480His) in the general population and its presence in three families with hearing loss, confirm the pathogenicity of this variant and illustrate the presentation of Perrault syndrome as nonsyndromic hearing loss in males and prepubertal females.

Aminoacyl-transfer RNA (tRNA) synthetases (aaRSs) are the largest protein family causatively linked to neurodegenerative Charcot-Marie-Tooth (CMT) disease. Dominant mutations cause the disease, and studies of CMT disease-causing mutant glycyl-tRNA synthetase (GlyRS) and tyrosyl-tRNA synthetase (TyrRS) showed their mutations create neomorphic structures consistent with a gain-of-function mechanism. In contrast, based on a haploid yeast model, loss of aminoacylation function was reported for CMT disease mutants in histidyl-tRNA synthetase (HisRS). However, neither that nor prior work of any CMT disease-causing aaRS investigated the aminoacylation status of tRNAs in the cellular milieu of actual patients. Using an assay that interrogated aminoacylation levels in patient cells, we investigated a HisRS-linked CMT disease family with the most severe disease phenotype. Strikingly, no difference in charged tRNA levels between normal and diseased family members was found. In confirmation, recombinant versions of 4 other HisRS CMT disease-causing mutants showed no correlation between activity loss in vitro and severity of phenotype in vivo. Indeed, a mutation having the most detrimental impact on activity was associated with a mild disease phenotype. In further work, using 3 independent biophysical analyses, structural opening (relaxation) of mutant HisRSs at the dimer interface best correlated with disease severity. In fact, the HisRS mutation in the severely afflicted patient family caused the largest degree of structural relaxation. These data suggest that HisRS-linked CMT disease arises from open conformation-induced mechanisms distinct from loss of aminoacylation., Competing Interests: Conflict of interest statement: T.W. is employed by the company Dynamic Biosensors GmbH.

The extra 5' guanine nucleotide (G-1) on tRNA His is a nearly universal feature that specifies tRNA His identity. The G-1 residue is either genome encoded or post-transcriptionally added by tRNA His guanylyltransferase (Thg1). Despite Caenorhabditis elegans being a Thg1-independent organism, its cytoplasmic tRNA His (CetRNA n His ) retains a genome-encoded G-1. Our study showed that this eukaryote possesses a histidyl-tRNA synthetase (CeHisRS) gene encoding two distinct HisRS isoforms that differ only at their N-termini. Most interestingly, its mitochondrial tRNA His (CetRNA m His ) lacks G-1, a scenario never observed in any organelle. This tRNA, while lacking the canonical identity element, can still be efficiently aminoacylated in vivo. Even so, addition of G-1 to CetRNA m His strongly enhanced its aminoacylation efficiency in vitro. Overexpression of CeHisRS successfully bypassed the requirement for yeast THG1 in the presence of CetRNA n His without G-1. Mutagenesis assays showed that the anticodon takes a primary role in CetRNA His identity recognition, being comparable to the universal identity element. Consequently, simultaneous introduction of both G-1 and the anticodon of tRNA His effectively converted a non-cognate tRNA to a tRNA His -like substrate. Our study suggests that a new balance between identity elements of tRNA His relieves HisRS from the absolute requirement for G-1.

Background: A 49-year-old male presented with late-onset demyelinating peripheral neuropathy, cerebellar atrophy, and cognitive deficit. Nerve biopsy revealed intra-axonal inclusions suggestive of polyglucosan bodies, raising the suspicion of adult polyglucosan bodies disease (OMIM 263570)., Methods and Results: While known genes associated with polyglucosan bodies storage were negative, whole-exome sequencing identified an unreported monoallelic variant, c.397G>T (p.Val133Phe), in the histidyl-tRNA synthetase ( HARS ) gene. While we did not identify mutations in genes known to be associated with polygucosan body disease, whole-exome sequencing revealed an unreported monoallelic variant, c.397G>T in the histidyl-tRNA synthetase (HARS) gene, encoding a substitution (Val133Phe) in the catalytic domain. Expression of this variant in patient cells resulted in reduced aminoacylation activity in extracts obtained from dermal fibroblasts, without compromising overall protein synthesis., Interpretation: Genetic variants in the genes coding for the different aminoacyl-tRNA synthases are associated with various clinical conditions. To date, a number of HARS variant have been associated with peripheral neuropathy, but not cognitive deficits. Further studies are needed to explore why HARS mutations confer a neuronal-specific phenotype., Competing Interests: The authors declare that they have no conflict of interest.

Histidyl-tRNA synthetase (HARS) ligates histidine to cognate tRNA molecules, which is required for protein translation. Mutations in HARS cause the dominant axonal peripheral neuropathy Charcot-Marie-Tooth disease type 2W (CMT2W); however, the precise molecular mechanism remains undefined. Here, we investigated three HARS missense mutations associated with CMT2W (p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly). The three mutations localize to the HARS catalytic domain and failed to complement deletion of the yeast ortholog (HTS1). Enzyme kinetics, differential scanning fluorimetry (DSF), and analytical ultracentrifugation (AUC) were employed to assess the effect of these substitutions on primary aminoacylation function and overall dimeric structure. Notably, the p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly HARS substitutions all led to reduced aminoacylation, providing a direct connection between CMT2W-linked HARS mutations and loss of canonical ARS function. While DSF assays revealed that only one of the variants (p.Val155Gly) was less thermally stable relative to wild-type, all three HARS mutants formed stable dimers, as measured by AUC. Our work represents the first biochemical analysis of CMT-associated HARS mutations and underscores how loss of the primary aminoacylation function can contribute to disease pathology., (© 2017 Wiley Periodicals, Inc.)

Pseudomonas aeruginosa histidyl-tRNA synthetase (HisRS) was selected as a target for antibiotic drug development. The HisRS protein was overexpressed in Escherichia coli and kinetically evaluated. The K M values for interaction of HisRS with its three substrates, histidine, ATP, and tRNA His , were 37.6, 298.5, and 1.5 μM, while the turnover numbers were 8.32, 16.8, and 0.57 s -1 , respectively. A robust screening assay was developed, and 800 natural products and 890 synthetic compounds were screened for inhibition of activity. Fifteen compounds with inhibitory activity were identified, and the minimum inhibitory concentration (MIC) was determined for each against a panel of nine pathogenic bacteria. Each compound exhibited broad-spectrum activity. Based on structural similarity and MIC results, four compounds, BT02C02, BT02D04, BT08E04, and BT09C11, were selected for additional analysis. These compounds inhibited the activity of HisRS with IC 50 values of 4.4, 9.7, 14.1, and 11.3 µM, respectively. Time-kill studies indicated a bacteriostatic mode of inhibition for each compound. BT02D04 and BT08E04 were noncompetitive with both histidine and ATP, BT02C02 was competitive with histidine but noncompetitive with ATP, and BT09C11 was uncompetitive with histidine and noncompetitive with ATP. These compounds were not observed to be toxic to human cell cultures.

Background: Patients with suspected idiopathic inflammatory myopathies (IIM) are commonly tested for the presence of anti-nuclear antibodies (ANA) by indirect immunofluorescence (IIF) on HEp-2 cell substrates. However, ANA-IIF false negative tests may occur in IIM because some antigens, such as Jo1 and Ro52, may be scarcely expressed on HEp-2 cells. In addition, cytoplasmic staining is often not appropriately investigated by a specific antibody assay, leading to decreased clinical sensitivity of the ANA test. We evaluated the diagnostic impact of different strategies using different combination of myositis-related autoantibody tests., Methods: Sera from 51 patients with an established diagnosis of IIM were tested for ANA by IIF on HEp-2 cells and for myositis-specific antibodies (MSA) and myositis-associated antibodies (MAA) by lineblot methods., Results: Forty-four/51 (86.3%) samples tested positive with at least one of the three methods and seven were negative with all methods. Of the 44 positive samples, 9 (20.5%) tested negative for the ANA-IIF test and positive for MAA/MSA. Anti-Ro52 were the most prevalent autoantibodies in IIM patients (21/51; 41%), frequently associated with anti-Jo1 antibodies (13/21; 62%). 13 (16%) anti-Ro52 and anti-Jo1 negative samples were reactive to MSA., Conclusions: Our findings suggest that when IIM is clinically suspected, the optimal diagnostic algorithm is to associate the ANA-IIF screening test with a specific test for anti-Ro52 and anti-Jo1 antibodies. Should all these tests be negative, serological tests for MSA are recommended., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Development of new aminoacyl-tRNA synthetase (aaRS)•tRNA pairs is central for incorporation of novel non-canonical amino acids (ncAAs) into proteins via genetic code expansion (GCE). The Escherichia coli and Caulobacter crescentus histidyl-tRNA synthetases (HisRS) evolved divergent mechanisms of tRNA His recognition that prevent their cross-reactivity. Although the E. coli HisRS•tRNA His pair is a good candidate for GCE, its use in C. crescentus is limited by the lack of established genetic selection methods and by the low transformation efficiency of C. crescentus., Methods: E. coli was genetically engineered to use a C. crescentus HisRS•tRNA His pair. Super-folder green fluorescent protein (sfGFP) and chloramphenicol acetyltransferase (CAT) were used as reporters for read-through assays. A library of 313 ncAAs coupled with the sfGFP reporter system was employed to investigate the specificity of E. coli HisRS in vivo., Results: A genomically modified E. coli strain (named MEOV1) was created. MEVO1 requires an active C. crescentus HisRS•tRNA His pair for growth, and displays a similar doubling time as the parental E. coli strain. sfGFP- and CAT-based assays showed that the E. coli HisRS•tRNA His pair is orthogonal in MEOV1 cells. A mutation in the anticodon loop of E. coli tRNA His CUA elevated its suppression efficiency by 2-fold., Conclusions: The C. crescentus HisRS•tRNA His pair functionally complements an E. coli ΔhisS strain. The E. coli HisRS•tRNA His is orthogonal in MEOV1 cells. E. coli tRNA His CUA is an efficient amber suppressor in MEOV1., General Significance: We developed a platform that allows protein engineering of E. coli HisRS that should facilitate GCE in E. coli. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Histidyl tRNA Synthetase (HARS) is a member of the aminoacyl tRNA synthetase (ARS) family of enzymes. This family of 20 enzymes is responsible for attaching specific amino acids to their cognate tRNA molecules, a critical step in protein synthesis. However, recent work highlighting a growing number of associations between ARS genes and diverse human diseases raises the possibility of new and unexpected functions in this ancient enzyme family. For example, mutations in HARS have been linked to two different neurological disorders, Usher Syndrome Type IIIB and Charcot Marie Tooth peripheral neuropathy. These connections raise the possibility of previously undiscovered roles for HARS in metazoan development, with alterations in these functions leading to complex diseases. In an attempt to establish Danio rerio as a model for studying HARS functions in human disease, we characterized the Danio rerio hars gene and compared it to that of human HARS. Using a combination of bioinformatics, molecular biology, and cellular approaches, we found that while the human genome encodes separate genes for cytoplasmic and mitochondrial HARS protein, the Danio rerio genome encodes a single hars gene which undergoes alternative splicing to produce the respective cytoplasmic and mitochondrial versions of Hars. Nevertheless, while the HARS genes of humans and Danio differ significantly at the genomic level, we found that they are still highly conserved at the amino acid level, underscoring the potential utility of Danio rerio as a model organism for investigating HARS function and its link to human diseases in vivo.

Histidyl-tRNA synthetase (HARS) is a highly conserved translation factor that plays an essential role in protein synthesis. HARS has been implicated in the human syndromes Charcot-Marie-Tooth (CMT) Type 2W and Type IIIB Usher (USH3B). The USH3B mutation, which encodes a Y454S substitution in HARS, is inherited in an autosomal recessive fashion and associated with childhood deafness, blindness, and episodic hallucinations during acute illness. The biochemical basis of the pathophysiologies linked to USH3B is currently unknown. Here, we present a detailed functional comparison of wild-type (WT) and Y454S HARS enzymes. Kinetic parameters for enzymes and canonical substrates were determined using both steady state and rapid kinetics. Enzyme stability was examined using differential scanning fluorimetry. Finally, enzyme functionality in a primary cell culture was assessed. Our results demonstrate that the Y454S substitution leaves HARS amino acid activation, aminoacylation, and tRNA His binding functions largely intact compared with those of WT HARS, and the mutant enzyme dimerizes like the wild type does. Interestingly, during our investigation, it was revealed that the kinetics of amino acid activation differs from that of the previously characterized bacterial HisRS. Despite the similar kinetics, differential scanning fluorimetry revealed that Y454S is less thermally stable than WT HARS, and cells from Y454S patients grown at elevated temperatures demonstrate diminished levels of protein synthesis compared to those of WT cells. The thermal sensitivity associated with the Y454S mutation represents a biochemical basis for understanding USH3B.

Differential scanning fluorimetry (DSF) is a fluorescence-based assay to evaluate protein stability by determining protein melting temperatures. Here, we describe the application of DSF to investigate aminoacyl-tRNA synthetase (AARS) stability and interaction with ligands. Employing three bacterial AARS enzymes as model systems, methods are presented here for the use of DSF to measure the apparent temperatures at which AARSs undergo melting transitions, and the effect of AARS substrates and inhibitors. One important observation is that the extent of temperature stability realized by an AARS in response to a particular bound ligand cannot be predicted a priori. The DSF method thus serves as a rapid and highly quantitative approach to measure AARS stability, and the ability of ligands to influence the temperature at which unfolding transitions occur., (Copyright © 2016. Published by Elsevier Inc.)

To determine how well DNA barcodes from the chloroplast region perform in forest dynamics plots (FDPs) from global CTFS-ForestGEO network, we analyzed DNA barcoding sequences of 1277 plant species from a wide phylogenetic range (3 FDPs in tropics, 5 in subtropics and 5 in temperate zone) and compared the rates of species discrimination (RSD). We quantified RSD by two DNA barcode combinations (rbcL + matK and rbcL + matK + trnH-psbA) using a monophyly-based method (GARLI). We defined two indexes of closely-related taxa (Gm/Gt and S/G ratios) and correlated these ratios with RSD. The combination of rbcL + matK averagely discriminated 88.65%, 83.84% and 72.51% at the local, regional and global scales, respectively. An additional locus trnH-psbA increased RSD by 2.87%, 1.49% and 3.58% correspondingly. RSD varied along a latitudinal gradient and were negatively correlated with ratios of closely-related taxa. Successes of species discrimination generally depend on scales in global FDPs. We suggested that the combination of rbcL + matK + trnH-psbA is currently applicable for DNA barcoding-based phylogenetic studies on forest communities.

Inherited peripheral neuropathies are a genetically heterogeneous group of disorders characterized by distal muscle weakness and sensory loss. Mutations in genes encoding aminoacyl-tRNA synthetases have been implicated in peripheral neuropathies, suggesting that these tRNA charging enzymes are uniquely important for the peripheral nerve. Recently, a mutation in histidyl-tRNA synthetase (HARS) was identified in a single patient with a late-onset, sensory-predominant peripheral neuropathy; however, the genetic evidence was lacking, making the significance of the finding unclear. Here, we present clinical, genetic, and functional data that implicate HARS mutations in inherited peripheral neuropathies. The associated phenotypic spectrum is broad and encompasses axonal and demyelinating motor and sensory neuropathies, including four young patients presenting with pure motor axonal neuropathy. Genome-wide linkage studies in combination with whole-exome and conventional sequencing revealed four distinct and previously unreported heterozygous HARS mutations segregating with autosomal dominant peripheral neuropathy in four unrelated families (p.Thr132Ile, p.Pro134His, p.Asp175Glu and p.Asp364Tyr). All mutations cause a loss of function in yeast complementation assays, and p.Asp364Tyr is dominantly neurotoxic in a Caenorhabditis elegans model. This study demonstrates the role of HARS mutations in peripheral neuropathy and expands the genetic and clinical spectrum of aminoacyl-tRNA synthetase-related human disease., (© The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Aminoacyl-tRNA synthetases (aaRSs) play a crucial role in protein translation by linking tRNAs with cognate amino acids. Among all the tRNAs, only tRNA(His) bears a guanine base at position -1 (G-1), and it serves as a major recognition element for histidyl-tRNA synthetase (HisRS). Despite strong interests in the histidylation mechanism, the tRNA recognition and aminoacylation details are not fully understood. We herein present the 2.55 Å crystal structure of HisRS complexed with tRNA(His), which reveals that G-1 recognition is principally nonspecific interactions on this base and is made possible by an enlarged binding pocket consisting of conserved glycines. The anticodon triplet makes additional specific contacts with the enzyme but the rest of the loop is flexible. Based on the crystallographic and biochemical studies, we inferred that the uniqueness of histidylation system originates from the enlarged binding pocket (for the extra base G-1) on HisRS absent in other aaRSs, and this structural complementarity between the 5' extremity of tRNA and enzyme is probably a result of coevolution of both., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd- phenotype), while other substitutions block kinase activation (Gcn- phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction.

As part of a project aimed at obtaining selective inhibitors and drug-like compounds targeting tRNA synthetases from trypanosomatids, we have elucidated the crystal structure of human cytosolic histidyl-tRNA synthetase (Hs-cHisRS) in complex with histidine in order to be able to compare human and parasite enzymes. The resultant structure of Hs-cHisRS•His represents the substrate-bound state (H-state) of the enzyme. It provides an interesting opportunity to compare with ligand-free and imidazole-bound structures Hs-cHisRS published recently, both of which represent the ligand-free state (F-state) of the enzyme. The H-state Hs-cHisRS undergoes conformational changes in active site residues and several conserved motif of HisRS, compared to F-state structures. The histidine forms eight hydrogen bonds with HisRS of which six engage the amino and carboxylate groups of this amino acid. The availability of published imidazole-bound structure provides a unique opportunity to dissect the structural roles of individual chemical groups of histidine. The analysis revealed the importance of the amino and carboxylate groups, of the histidine in leading to these dramatic conformational changes of the H-state. Further, comparison with previously published trypanosomatid HisRS structures reveals a pocket in the F-state of the parasite enzyme that may provide opportunities for developing specific inhibitors of Trypanosoma brucei HisRS., (Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.)

Inflammatory and debilitating myositis and interstitial lung disease are commonly associated with autoantibodies (anti-Jo-1 antibodies) to cytoplasmic histidyl-tRNA synthetase (HisRS). Anti-Jo-1 antibodies from different disease-afflicted patients react mostly with spatially separated epitopes in the three-dimensional structure of human HisRS. We noted that two HisRS splice variants (SVs) include these spatially separated regions, but each SV lacks the HisRS catalytic domain. Despite the large deletions, the two SVs cross-react with a substantial population of anti-Jo-l antibodies from myositis patients. Moreover, expression of at least one of the SVs is up-regulated in dermatomyositis patients, and cell-based experiments show that both SVs and HisRS can be secreted. We suggest that, in patients with inflammatory myositis, anti-Jo-1 antibodies may have extracellular activity., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

We tested the idea that ancestral class I and II aminoacyl-tRNA synthetases arose on opposite strands of the same gene. We assembled excerpted 94-residue Urgenes for class I tryptophanyl-tRNA synthetase (TrpRS) and class II Histidyl-tRNA synthetase (HisRS) from a diverse group of species, by identifying and catenating three blocks coding for secondary structures that position the most highly conserved, active-site residues. The codon middle-base pairing frequency was 0.35 ± 0.0002 in all-by-all sense/antisense alignments for 211 TrpRS and 207 HisRS sequences, compared with frequencies between 0.22 ± 0.0009 and 0.27 ± 0.0005 for eight different representations of the null hypothesis. Clustering algorithms demonstrate further that profiles of middle-base pairing in the synthetase antisense alignments are correlated along the sequences from one species-pair to another, whereas this is not the case for similar operations on sets representing the null hypothesis. Most probable reconstructed sequences for ancestral nodes of maximum likelihood trees show that middle-base pairing frequency increases to approximately 0.42 ± 0.002 as bacterial trees approach their roots; ancestral nodes from trees including archaeal sequences show a less pronounced increase. Thus, contemporary and reconstructed sequences all validate important bioinformatic predictions based on descent from opposite strands of the same ancestral gene. They further provide novel evidence for the hypothesis that bacteria lie closer than archaea to the origin of translation. Moreover, the inverse polarity of genetic coding, together with a priori α-helix propensities suggest that in-frame coding on opposite strands leads to similar secondary structures with opposite polarity, as observed in TrpRS and HisRS crystal structures.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes responsible for ligating amino acids to cognate tRNA molecules. Mutations in four genes encoding an ARS have been implicated in inherited peripheral neuropathy with an axonal pathology, suggesting that all ARS genes are relevant candidates for disease in patients with related phenotypes. Here, we present results from a mutation screen of the histidyl-tRNA synthetase (HARS) gene in a large cohort of patients with peripheral neuropathy. These efforts revealed a rare missense variant (c.410G>A/p.Arg137Gln) that resides at a highly conserved amino acid, represents a loss-of-function allele when evaluated in yeast complementation assays, and is toxic to neurons when expressed in a worm model. In addition to the patient with peripheral neuropathy, p.Arg137Gln HARS was detected in three individuals by genome-wide exome sequencing. These findings suggest that HARS is the fifth ARS locus associated with axonal peripheral neuropathy. Implications for identifying ARS alleles in human populations and assessing them for a role in neurodegenerative phenotypes are discussed., (© 2012 Wiley Periodicals, Inc.)

Aminoacyl-tRNA synthetases (AARSs) catalyze aminoacylation of tRNAs in the cytoplasm. Surprisingly, AARSs also have critical extracellular and nuclear functions. Evolutionary pressure for new functions might be manifested by splice variants that skip only an internal catalytic domain (CD) and link noncatalytic N- and C-terminal polypeptides. Using disease-associated histidyl-tRNA synthetase (HisRS) as an example, we found an expressed 171-amino acid protein (HisRSΔCD) that deleted the entire CD, and joined an N-terminal WHEP to the C-terminal anticodon-binding domain (ABD). X-ray crystallography and three-dimensional NMR revealed the structures of human HisRS and HisRSΔCD. In contrast to homodimeric HisRS, HisRSΔCD is monomeric, where rupture of the ABD's packing with CD resulted in a dumbbell-like structure of flexibly linked WHEP and ABD domains. In addition, the ABD of HisRSΔCD presents a distinct local conformation. This natural internally deleted HisRS suggests evolutionary pressure to reshape AARS tertiary and quaternary structures for repurposing., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In polymyositis and inclusion body myositis, muscle fibers are surrounded and invaded by CD8-positive cytotoxic T cells expressing the αβ-T cell receptor (αβ-TCR) for antigen. In a rare variant of myositis, muscle fibers are similarly attacked by CD8-negative T cells expressing the γδ-TCR (γδ-T cell-mediated myositis). We investigated the antigen specificity of a human γδ-TCR previously identified in an autoimmune tissue lesion of γδ-T cell-mediated myositis. We show that this Vγ1.3Vδ2-TCR, termed M88, recognizes various proteins from different species. Several of these proteins belong to the translational apparatus, including some bacterial and human aminoacyl-tRNA synthetases (AA-RS). Specifically, M88 recognizes histidyl-tRNA synthetase, an antigen known to be also targeted by autoantibodies called anti-Jo-1. The M88 target epitope is strictly conformational, independent of post-translational modification, and exposed on the surface of the respective antigenic protein. Extensive mutagenesis of the translation initiation factor-1 from Escherichia coli (EcIF1), which served as a paradigm antigen with known structure, showed that a short α-helical loop around amino acids 39 to 42 of EcIF1 is a major part of the M88 epitope. Mutagenesis of M88 showed that the complementarity determining regions 3 of both γδ-TCR chains contribute to antigen recognition. M88 is the only known example of a molecularly characterized γδ-TCR expressed by autoaggressive T cells in tissue. The observation that AA-RS are targeted by a γδ-T cell and by autoantibodies reveals an unexpected link between T cell and antibody responses in autoimmune myositis.

Perrault syndrome is a genetically heterogeneous recessive disorder characterized by ovarian dysgenesis and sensorineural hearing loss. In a nonconsanguineous family with five affected siblings, linkage analysis and genomic sequencing revealed the genetic basis of Perrault syndrome to be compound heterozygosity for mutations in the mitochondrial histidyl tRNA synthetase HARS2 at two highly conserved amino acids, L200V and V368L. The nucleotide substitution creating HARS2 p.L200V also created an alternate splice leading to deletion of 12 codons from the HARS2 message. Affected family members thus carried three mutant HARS2 transcripts. Aminoacylation activity of HARS2 p.V368L and HARS2 p.L200V was reduced and the deletion mutant was not stably expressed in mammalian mitochondria. In yeast, lethality of deletion of the single essential histydyl tRNA synthetase HTS1 was fully rescued by wild-type HTS1 and by HTS1 p.L198V (orthologous to HARS2 p.L200V), partially rescued by HTS1 p.V381L (orthologous to HARS2 p.V368L), and not rescued by the deletion mutant. In Caenorhabditis elegans, reduced expression by RNAi of the single essential histydyl tRNA synthetase hars-1 severely compromised fertility. Together, these data suggest that Perrault syndrome in this family was caused by reduction of HARS2 activity. These results implicate aberrations of mitochondrial translation in mammalian gonadal dysgenesis. More generally, the relationship between HARS2 and Perrault syndrome illustrates how causality may be demonstrated for extremely rare inherited mutations in essential, highly conserved genes.

Background: Pelobacter carbinolicus, a bacterium of the family Geobacteraceae, cannot reduce Fe(III) directly or produce electricity like its relatives. How P. carbinolicus evolved is an intriguing problem. The genome of P. carbinolicus contains clustered regularly interspaced short palindromic repeats (CRISPR) separated by unique spacer sequences, which recent studies have shown to produce RNA molecules that interfere with genes containing identical sequences., Results: CRISPR spacer #1, which matches a sequence within hisS, the histidyl-tRNA synthetase gene of P. carbinolicus, was shown to be expressed. Phylogenetic analysis and genetics demonstrated that a gene paralogous to hisS in the genomes of Geobacteraceae is unlikely to compensate for interference with hisS. Spacer #1 inhibited growth of a transgenic strain of Geobacter sulfurreducens in which the native hisS was replaced with that of P. carbinolicus. The prediction that interference with hisS would result in an attenuated histidyl-tRNA pool insufficient for translation of proteins with multiple closely spaced histidines, predisposing them to mutation and elimination during evolution, was investigated by comparative genomics of P. carbinolicus and related species. Several ancestral genes with high histidine demand have been lost or modified in the P. carbinolicus lineage, providing an explanation for its physiological differences from other Geobacteraceae., Conclusions: The disappearance of multiheme c-type cytochromes and other genes typical of a metal-respiring ancestor from the P. carbinolicus lineage may be the consequence of spacer #1 interfering with hisS, a condition that can be reproduced in a heterologous host. This is the first successful co-introduction of an active CRISPR spacer and its target in the same cell, the first application of a chimeric CRISPR construct consisting of a spacer from one species in the context of repeats of another species, and the first report of a potential impact of CRISPR on genome-scale evolution by interference with an essential gene.

Nearly all tRNA(His) species have an additional 5' guanine nucleotide (G(-1)). G(-1) is encoded opposite C(73) in nearly all prokaryotes and in some archaea, and is added post-transcriptionally by tRNA(His) guanylyltransferase (Thg1) opposite A(73) in eukaryotes, and opposite C(73) in other archaea. These divergent mechanisms of G(-1) conservation suggest that G(-1) might have an important cellular role, distinct from its role in tRNA(His) charging. Thg1 is also highly conserved and is essential in the yeast Saccharomyces cerevisiae. However, the essential roles of Thg1 are unclear since Thg1 also interacts with Orc2 of the origin recognition complex, is implicated in the cell cycle, and catalyzes an unusual template-dependent 3'-5' (reverse) polymerization in vitro at the 5' end of activated tRNAs. Here we show that thg1-Delta strains are viable, but only if histidyl-tRNA synthetase and tRNA(His) are overproduced, demonstrating that the only essential role of Thg1 is its G(-1) addition activity. Since these thg1-Delta strains have severe growth defects if cytoplasmic tRNA(His) A(73) is overexpressed, and distinct, but milder growth defects, if tRNA(His) C(73) is overexpressed, these results show that the tRNA(His) G(-1) residue is important, but not absolutely essential, despite its widespread conservation. We also show that Thg1 catalyzes 3'-5' polymerization in vivo on tRNA(His) C(73), but not on tRNA(His) A(73), demonstrating that the 3'-5' polymerase activity is pronounced enough to have a biological role, and suggesting that eukaryotes may have evolved to have cytoplasmic tRNA(His) with A(73), rather than C(73), to prevent the possibility of 3'-5' polymerization.

Aims: To elucidate the detailed mechanism of histamine production by Photobacterium damselae subsp. damselae., Methods and Results: Histidine decarboxylase and related genes of P. damselae subsp. damselae were cloned, and three open reading frames named as hdcT, hdcA and hisRS were identified. The hdcA gene encodes a polypeptide of 377 amino acids and is considered to be the pyridoxal-P dependent histidine decarboxylase. The hdcT gene is assumed to be a histidine/histamine antiporter, and the hisRS gene is considered to be a histidyl-tRNA synthetase. Recombinant Escherichia coli strains harbouring plasmids carrying the P. damselae hdc genes were shown to over-excrete histamine extracellularly. Northern blot analysis and quantitative RT-PCR revealed high levels of mono- and bi-cistronic transcripts of hdcA, hdcT and hisRS genes under conditions of low pH and histidine excess., Conclusions: The hdcA gene of P. damselae was constructed as an operon with putative histidine/histamine antiporter and histidyl-tRNA synthetase. Mono- and poly-cistronic transcripts and acid induction were detected., Significance and Impact of the Study: This is the first report of cloning the histidine decarboxylase gene cluster in gram-negative bacteria. Also, these genes were induced under acidic conditions and in the presence of excess histidine.

Aim: To obtain highly purified Jo-1 autoantigens., Methods: The full length of DNA sequence coding for Jo-1 (histidyl-tRNA synthetase) was obtained from human placenta by RT-PCR and then it was inserted into pTYB11 or pMAL-c to construct the expression vectors pTYB11-Jo-1 and pMAL-c-Jo-1. The recombinant plasmids were transformed into ER2566 and BL21 of E.coli, respectively., Results: The fusion Jo-1 antigens were expressed, Western blot analysis demonstrated they responded specifically to anti-Jo-1 antibody from the patients with autoimmune disease polymyositis and dermatomyositis, but did not respond to normal sera and 188 sera containing anti-RNP, Sm, Ro/La or RNP/Ro antibodies from rheumatosis patients., Conclusion: The expressed protein of pMAL-c-Jo-1 is soluble, which accounts for more then 50% of total proteins of cells and can be purified by affinity chromatography. The purified proteins can be used as reagents for determining the anti Jo-1 antibody in the serum of patients.

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.

Egr1 (Zif268) is an immediate early gene encoding an inducible transcription factor involved in synaptic plasticity and several forms of memory in rodents. Using 2-DE and MS, we compared proteomes of hippocampal subregions and cortex in Egr1-deficient and wild-type littermates. Two significant differences were identified: a shift in the pI of the molecular chaperone mortalin (mtHsp70/PBP74/Grp75) and the apparent disappearance of histidyl tRNA synthetase (HisRS). We found that the pI shift for mortalin in Egr1-deficient mice was caused by a difference in protein sequence: D626G. Using cDNA sequencing, we demonstrated for both mortalin and HisRS that protein differences were not due to a lack of Egr1 but to DNA polymorphism between the C57Bl/6J and 129/Sv strains used to generate the Egr1-deficient mice. Our results show that mortalin and HisRS genes, which map closely to the Egr1 locus, have conserved the 129/Sv haplotype despite numerous back-crossing of the null mice progeny with C57Bl/6J animals. This demonstrates that allelic differences between mouse strains can introduce variations in differential proteomic analyses of genetically modified organisms. Finally, we report the identification of new isoforms of HisRS and mortalin (mot-3) encoded by the 129/Sv haplotype.

Autoantibodies against aminoacyl-tRNA synthetases (anti-AARS) belong to the group of the myositis specific autoantibodies (MSAs). Their association with the onset and development of the idiopathic inflammatory myopathies (IIM) implies their participation in the pathogenesis of the diseases. Since the appearing of anti-Jo-1 and other anti-AARSs is related to characteristic immunogenetic and clinical features, they can be considered specific markers in diagnosis and classification of patients affected by IIM. Here, we present an overview of anti-AARSs, their chemoattractant properties, their detection methods, genetic risks, and protective factors.

Objective: To investigate possible associations of HLA polymorphisms with idiopathic inflammatory myopathy (IIM) in African Americans, and to compare this with HLA associations in European American IIM patients with IIM., Methods: Molecular genetic analyses of HLA-A, B, Cw, DRB1, and DQA1 polymorphisms were performed in a large population of African American patients with IIM (n = 262) in whom the major clinical and autoantibody subgroups were represented. These data were compared with similar information previously obtained from European American patients with IIM (n = 571)., Results: In contrast to European American patients with IIM, African American patients with IIM, in particular those with polymyositis, had no strong disease associations with HLA alleles of the 8.1 ancestral haplotype; however, African Americans with dermatomyositis or with anti-Jo-1 autoantibodies shared the risk factor HLA-DRB1*0301 with European Americans. We detected novel HLA risk factors in African American patients with myositis overlap (DRB1*08) and in African American patients producing anti-signal recognition particle (DQA1*0102) and anti-Mi-2 autoantibodies (DRB1*0302). DRB1*0302 and the European American-, anti-Mi-2-associated risk factor DRB1*0701 were found to share a 4-amino-acid sequence motif, which was predicted by comparative homology analyses to have identical 3-dimensional orientations within the peptide-binding groove., Conclusion: These data demonstrate that North American IIM patients from different ethnic groups have both shared and distinct immunogenetic susceptibility factors, depending on the clinical phenotype. These findings, obtained from the largest cohort of North American minority patients with IIM studied to date, add additional support to the hypothesis that the myositis syndromes comprise multiple, distinct disease entities, perhaps arising from divergent pathogenic mechanisms and/or different gene-environment interactions.

We present TFAM, an automated, statistical method to classify the identity of tRNAs. TFAM, currently optimized for bacteria, classifies initiator tRNAs and predicts the charging identity of both typical and atypical tRNAs such as suppressors with high confidence. We show statistical evidence for extensive variation in tRNA identity determinants among bacterial genomes due to variation in overall tDNA base content. With TFAM we have detected the first case of eukaryotic-like tRNA identity rules in bacteria. An alpha-proteobacterial clade encompassing Rhizobiales, Caulobacter crescentus and Silicibacter pomeroyi, unlike a sister clade containing the Rickettsiales, Zymomonas mobilis and Gluconobacter oxydans, uses the eukaryotic identity element A73 instead of the highly conserved prokaryotic element C73. We confirm divergence of bacterial histidylation rules by demonstrating perfect covariation of alpha-proteobacterial tRNA(His) acceptor stems and residues in the motif IIb tRNA-binding pocket of their histidyl-tRNA synthetases (HisRS). Phylogenomic analysis supports lateral transfer of a eukaryotic-like HisRS into the alpha-proteobacteria followed by in situ adaptation of the bacterial tDNA(His) and identity rule divergence. Our results demonstrate that TFAM is an effective tool for the bioinformatics, comparative genomics and evolutionary study of tRNA identity.

The amplification of an internal fragment of the hdcA gene for histidine decarboxylase in Lactobacillus buchneri showed the gene to be located on the bacterial chromosome. Reverse PCR was then used to amplify both it and its adjacent genes. The histidine decarboxylase cluster was found to be composed of four genes: hdcC (expressed in Lactococcus lactis, the product of which is located in the membrane, suggesting it to be a histidine/histamine antiporter), hdcA (which encodes histidine decarboxylase), hdcB (of unknown function but co-transcribed as bicistronic mRNA together with hdcA) and hisS (the only copy of a gene encoding a histidyl-tRNA synthetase in Lb. buchneri). The expression of hisS depends on the histidine concentration of the growth medium, and it can be transcribed as monocistronic or hdcA-hdcB-hisS polycistronic mRNA.

El The Jo-1 syndrome is an autoimmune disease which is characterized by the presence of autoantibodies against the Jo-1 antigen. The designation Jo-1 is derived from the name of the first patient (John P.) who was tested positive for this antibody. This patient suffered from polymyositis and fibrosing alveolitis. The Jo-1 antigen was identified as histidyl-transfer-RNA synthetase present in the cytosol. The Jo-1 syndrome is a member of a family of autoimmune diseases, called anti-synthetase syndromes. These syndromes are characterized by autoantibodies directed against aminoacyl-transfer-RNA synthetases. The etiology of the Jo-1 syndrome is unknown. The most frequent clinical manifestation is myositis, which may present as polymyositis or dermatomyositis. In addition to muscle involvement, interstitial lung disease is frequently found and critical for the prognosis. Furthermore, symptoms of other autoimmune disorders such as polyarthritis may occur. Similar to polymyositis and dermatomyositis, the Jo-1 syndrome may present as myositis overlap syndrome. In these cases, antibodies against U1-RNP are detected. The Jo-1 syndrome responds to treatment with corticosteroids and, if necessary, azathioprine, methotrexate or cyclophosphamide. The clinical manifestations of the Jo-1 syndrome are illustrated by two clinical cases.

Aminoacyl-tRNA synthetases (aaRS) join amino acids to their cognate transfer RNAs, establishing an essential coding relationship in translation. To investigate the mechanism of aminoacyl transfer in class II Escherichia coli histidyl-tRNA synthetase (HisRS), we devised a rapid quench assay. Under single turnover conditions with limiting tRNA, aminoacyl transfer proceeds at 18.8 s(-)(1), whereas in the steady state, the overall rate of aminoacylation is limited by amino acid activation to a rate of 3 s(-)(1). In vivo, this mechanism may serve to allow the size of amino acid pools and energy charge to control the rate of aminoacylation and thus protein synthesis. Aminoacyl transfer experiments using HisRS active site mutants and phosphorothioate-substituted adenylate showed that substitution of the nonbridging Sp oxygen of the adenylate decreased the transfer rate at least 10 000-fold, providing direct experimental evidence for the role of this group as a general base for the reaction. Other kinetic experiments revealed that the rate of aminoacyl transfer is independent of the interaction between the carboxyamide group of Gln127 and the alpha-carboxylate carbon, arguing against the formation of a tetrahedral intermediate during the aminoacyl transfer. These experiments support a substrate-assisted concerted mechanism for HisRS, a feature that may generalize to other aaRS, as well as the peptidyl transferase center.

Aminoacylation of a transfer RNA (tRNA) by its cognate aminoacyl-tRNA synthetase relies upon the recognition of specific nucleotides as well as conformational features within the tRNA by the synthetase. In Escherichia coli, the aminoacylation of tRNA(His) by histidyl-tRNA synthetase (HisRS) is highly dependent upon the recognition of the unique G-1:C73 base pair and the 5'-monophosphate. This work investigates the RNA-protein interactions between the HisRS active site and these critical recognition elements. A homology model of the tRNA(His)-HisRS complex was generated and used to design site-specific mutants of possible G-1:C73 contacts. Aminoacylation assays were performed with these HisRS mutants and N-1:C73 tRNA(His) and microhelix(His) variants. Complete suppression of the negative effect of 5'-phosphate deletion by R123A HisRS, as well as the increased discrimination of Q118E HisRS against a 5'-triphosphate, suggests a possible interaction between the 5'-phosphate and active-site residues Arg123 and Gln118 in which these residues create a sterically and electrostatically favorable pocket for the binding of the negatively charged phosphate group. Additionally, a network of interactions appears likely between G-1 and Arg116, Arg123, and Gln118 because mutation of these residues significantly reduced the sensitivity of HisRS to changes at G-1. Our studies also support an interaction previously proposed between Gln118 and C73. Defining the RNA-protein interactions critical for efficient aminoacylation by E. coli HisRS helps to further characterize the active site of this enzyme and improves our understanding of how the unique identity elements in the acceptor stem of tRNA(His) confer specificity.

Background: RBCs of the Hy- phenotype have, in the past, been typed as Gy(a+w), Hy-, Jo(a-), and RBCs with the Jo(a-) phenotype type Gy(a+), Hy+w, and Jo(a-). Anti-Hy and anti-Joa are difficult to identify mainly because appropriate reagent RBCs are poorly characterized. Historically, anti-Joa has not reacted with RBCs with either phenotype. This report describes a case of an anti-Joa that shows Hy- RBCs express some Joa antigen, albeit weakly., Case Report: Anti-Joa was identified in a serum sample of a 71-year-old woman. The antibody reacted 1+ to 2+ by the IAT with all untreated and ficin-treated panel RBCs and did not react with Gy(a-) RBCs and Jo(a-) RBCs. Unexpectedly, the serum sample reacted weakly with six of eight RBC samples with the Hy- phenotype. The anti-Joa was adsorbed onto and eluted from Hy- RBCs, indicating the presence of weak Joa antigen. The patient's RBCs typed Gy(a+), Hy+, Jo(a-). DNA studies using PCR-RFLP analysis showed the patient to be homozygous for the JO allele, which is consistent with the serologically determined Jo(a-) status., Conclusion: The DNA and serologic evidence of this case show that Hy- RBCs may express low levels of Joa antigen, which contradicts previously published data concerning the Joa type of Hy- RBCs.

The aim of this study was to express and purify human histydyl-tRNA synthetase related gene and to prepare its polyantibody. The open reading frame was amplified by PCR, and then recombined into prokaryotic expression vector pQE30 and transformed into E. coli M15 for expression. The expressed products were induced by IPTG after the reconstructed pQE30 was transferred into M15. After purified by Ni affinity chromatography, the product was identified to be a single band by SDS-PAGE. The rabbits were inoculated with purified products. High-titer polyantibody was successfully prepared. Highly-purified expression product and prepared polyantibody may provide a good basis for further study.

A 1209 bp novel cDNA with a putative open reading frame of 627 bp that has an overlapped fragment of 527 bp with FKBP25 was isolated from a human fetal brain library. Northern Blot analysis detected a signal about 1.5 kb that suggests it is a full-length cDNA. Reverse transcription-PCR demonstrates that the transcripts are high in adult brain, testis, ovary, spleen, and fetal brain. Comparing the cDNA with the human genome database identifies a contig that reveals that the gene contains six exons and is located on human chromosome 20p11. The gene encodes a putative protein with an unknown domain conserved in many species and is similar to bacterial histidyl-tRNA synthetase, and so it is named human histidyl-tRNA-synthetase-related gene.

Histidyl-tRNA synthetase catalyses the covalent ligation of histidine to its cognate tRNA as an early step in protein biosynthesis. In humans, the histidyl-tRNA synthetase gene (HARS) is oriented opposite of a synthetase-like gene (HARSL) that bears striking homology to HARS. In this report, we describe the genomic organization of the HARS/HARSL locus and map multiple transcripts originating from a bi-directional promoter controlling the differential expression of these genes. The HARS and HARSL genes each contain 13 exons with strong structural and sequence homology over exons 3-12. HARS transcripts originate from two distinct promoters; a cluster of short transcripts map 15-65 bp upstream of the HARS ORF while a single, longer transcript (352 bp 5(')-UTR) maps to a distal promoter. Similarly, multiple HARSL transcripts (mapping 10-198 bp upstream of its ORF) are produced by the shared bi-directional promoter. Human and rodent HARS/HARSL loci are homologous and support a model of inverted gene duplication to explain the emergence of HARSL during mammalian evolution.

The protein kinase GCN2 is activated in amino acid-starved cells on binding of uncharged tRNA to a histidyl-tRNA synthetase (HisRS)-related domain. We isolated two point mutations in the protein kinase (PK) domain, R794G and F842L, that permit strong kinase activity in the absence of tRNA binding. These mutations also bypass the requirement for ribosome binding, dimerization, and association with the GCN1/GCN20 regulatory complex, suggesting that all of these functions facilitate tRNA binding to wild-type GCN2. While the isolated wild-type PK domain was completely inert, the mutant PK was highly active in vivo and in vitro. These results identify an inhibitory structure intrinsic to the PK domain that must be overcome on tRNA binding by interactions with a regulatory region, most likely the N terminus of the HisRS segment. As Arg 794 and Phe 842 are predicted to lie close to one another and to the active site, they may participate directly in misaligning active site residues. Autophosphorylation of the activation loop was stimulated by R794G and F842L, and the autophosphorylation sites remained critical for GCN2 function in the presence of these mutations. Our results imply a two-step activation mechanism involving distinct conformational changes in the PK domain.

Aminoacyl-tRNA synthetases (ARSs) ligate amino acids to their cognate tRNAs. It has been suggested that mammalian ARSs are linked to the EF-1 complex for efficient channeling of aminoacyl tRNAs to ribosome. Here we systemically investigated possible interactions between human ARSs and the subunits of EF-1 (alpha, beta, gamma, and delta) using a yeast two-hybrid assay. Among the 80 tested pairs, leucyl- and histidyl-tRNA synthetases were found to make strong and specific interaction with the EF-1gamma and beta while glu-proly-, glutaminyl-, alanyl-, aspartyl-, lysyl-, phenylalanyl-, glycyl-, and tryptophanyl-tRNA synthetases showed moderate interactions with the different EF-1 subunits. The interactions of leucyl- and histidyl-tRNA synthetase with the EF-1 complex were confirmed by immunoprecipitation and in vitro pull-down experiments. Interestingly, the aminoacylation activities of these two enzymes, but not other ARSs, were stimulated by the cofactor of EF-1, GTP. These data suggest that a systematic interaction network may exist between mammalian ARSs and EF-1 subunits probably to enhance the efficiency of in vivo protein synthesis., (©2002 Elsevier Science (USA).)

GCN2 stimulates translation of GCN4 mRNA in amino acid-starved cells by phosphorylating translation initiation factor 2. GCN2 is activated by binding of uncharged tRNA to a domain related to histidyl-tRNA synthetase (HisRS). The HisRS-like region contains two dimerization domains (HisRS-N and HisRS-C) required for GCN2 function in vivo but dispensable for dimerization by full-length GCN2. Residues corresponding to amino acids at the dimer interface of Escherichia coli HisRS were required for dimerization of recombinant HisRS-N and for tRNA binding by full-length GCN2, suggesting that HisRS-N dimerization promotes tRNA binding and kinase activation. HisRS-N also interacted with the protein kinase (PK) domain, and a deletion impairing this interaction destroyed GCN2 function without reducing tRNA binding; thus, HisRS-N-PK interaction appears to stimulate PK function. The C-terminal domain of GCN2 (C-term) interacted with the PK domain in a manner disrupted by an activating PK mutation (E803V). These results suggest that the C-term is an autoinhibitory domain, counteracted by tRNA binding. We conclude that multiple domain interactions, positive and negative, mediate the activation of GCN2 by uncharged tRNA.

Transfer RNA (tRNA) identity determinants help preserve the specificity of aminoacylation in vivo, and prevent cross-species interactions. Here, we investigate covariation between the discriminator base (N73) element in histidine tRNAs and residues in the histidyl-tRNA synthetase (HisRS) motif 2 loop. A model of the Escherichia coli HisRS--tRNA(His) complex predicts an interaction between the prokaryotic conserved glutamine 118 of the motif 2 loop and cytosine 73. The substitution of Gln 118 in motif 2 with glutamate decreased discrimination between cytosine and uracil some 50-fold, but left overall rates of adenylation and aminoacylation unaffected. By contrast, substitutions at neighboring Glu 115 and Arg 121 affected both adenylation and aminoacylation, consistent with their predicted involvement in both half-reactions. Additional evidence for the involvement of the motif 2 loop was provided by functional analysis of a hybrid Saccharomyces cerevisiae-- E. coli HisRS possessing the 11 amino acid motif 2 loop of the yeast enzyme. Despite an overall decreased activity of nearly 1000-fold relative to the E. coli enzyme, the chimera nevertheless exhibited a modest preference for the yeast tRNA(His) over the E. coli tRNA, and preferred wild-type yeast tRNA(His) to a variant with C at the discriminator position. These experiments suggest that part of, but not all of, the specificity is provided by the motif 2 loop. The close interaction between enzyme loop and RNA sequence elements suggested by these experiments reflects a covariation between enzyme and tRNA that may have acted to preserve aminoacylation fidelity over evolutionary time.