Towards a Cure for HARS Disease.

Histidyl-tRNA synthetase (HARS) ligates histidine to its cognate transfer RNA (tRNAHis). 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.

Neuropathy-associated histidyl-tRNA synthetase variants attenuate protein synthesis in vitro and disrupt axon outgrowth in developing zebrafish.

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.

Rational Design of Aptamer-Tagged tRNAs.

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.

Plant-Specific Domains and Fragmented Sequences Imply Non-Canonical Functions in Plant Aminoacyl-tRNA Synthetases.

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.

Bi-allelic mutations in HARS1 severely impair histidyl-tRNA synthetase expression and enzymatic activity causing a novel multisystem ataxic syndrome.

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.

A recurrent missense variant in HARS2 results in variable sensorineural hearing loss in three unrelated families.

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.

CMT disease severity correlates with mutation-induced open conformation of histidyl-tRNA synthetase, not aminoacylation loss, in patient cells.

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.

Naturally occurring dual recognition of tRNAHis substrates with and without a universal identity element.

The extra 5' guanine nucleotide (G-1) on tRNAHis is a nearly universal feature that specifies tRNAHis identity. The G-1 residue is either genome encoded or post-transcriptionally added by tRNAHis guanylyltransferase (Thg1). Despite Caenorhabditis elegans being a Thg1-independent organism, its cytoplasmic tRNAHis (CetRNAnHis) 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 tRNAHis (CetRNAmHis) 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 CetRNAmHis strongly enhanced its aminoacylation efficiency in vitro. Overexpression of CeHisRS successfully bypassed the requirement for yeast THG1 in the presence of CetRNAnHis without G-1. Mutagenesis assays showed that the anticodon takes a primary role in CetRNAHis identity recognition, being comparable to the universal identity element. Consequently, simultaneous introduction of both G-1 and the anticodon of tRNAHis effectively converted a non-cognate tRNA to a tRNAHis-like substrate. Our study suggests that a new balance between identity elements of tRNAHis relieves HisRS from the absolute requirement for G-1.

Peripheral neuropathy and cognitive impairment associated with a novel monoallelic HARS variant.

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.

Identification of Chemical Compounds That Inhibit the Function of Histidyl-tRNA Synthetase from Pseudomonas aeruginosa.

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 KM values for interaction of HisRS with its three substrates, histidine, ATP, and tRNAHis, 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 IC50 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.

Substrate interaction defects in histidyl-tRNA synthetase linked to dominant axonal peripheral neuropathy.

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.

An effective algorithm for the serological diagnosis of idiopathic inflammatory myopathies: The key role of anti-Ro52 antibodies.

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.

A single Danio rerio hars gene encodes both cytoplasmic and mitochondrial histidyl-tRNA synthetases.

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.

The Usher Syndrome Type IIIB Histidyl-tRNA Synthetase Mutation Confers Temperature Sensitivity.

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 tRNAHis 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.

A genomically modified Escherichia coli strain carrying an orthogonal E. coli histidyl-tRNA synthetase•tRNAHis pair.

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 tRNAHis recognition that prevent their cross-reactivity. Although the E. coli HisRS•tRNAHis 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•tRNAHis 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•tRNAHis 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•tRNAHis pair is orthogonal in MEOV1 cells. A mutation in the anticodon loop of E. coli tRNAHisCUA elevated its suppression efficiency by 2-fold. CONCLUSIONS: The C. crescentus HisRS•tRNAHis pair functionally complements an E. coli ΔhisS strain. The E. coli HisRS•tRNAHis is orthogonal in MEOV1 cells. E. coli tRNAHisCUA 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.

Characterization of aminoacyl-tRNA synthetase stability and substrate interaction by differential scanning fluorimetry.

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.

Closely-related taxa influence woody species discrimination via DNA barcoding: evidence from global forest dynamics plots.

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.

Loss of function mutations in HARS cause a spectrum of inherited peripheral neuropathies.

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.

Structural basis for recognition of G-1-containing tRNA by histidyl-tRNA synthetase.

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.

Interaction between the tRNA-binding and C-terminal domains of Yeast Gcn2 regulates kinase activity in vivo.

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.