Aminoacyl Transfer Ribonucleic Acid Synthetases Research Articles

Investigation in yeast of novel variants in mitochondrial aminoacyl-tRNA synthetases WARS2, NARS2, and RARS2 genes associated with mitochondrial diseases.

Aminoacyl-transfer RiboNucleic Acid synthetases (ARSs) are essential enzymes that catalyze the attachment of each amino acid to their cognate tRNAs. Mitochondrial ARSs (mtARSs), which ensure protein synthesis within the mitochondria, are encoded by nuclear genes and imported into the organelle after translation in the cytosol. The extensive use of next generation sequencing (NGS) has resulted in an increasing number of variants in mtARS genes being identified and associated with mitochondrial diseases. The similarities between yeast and human mitochondrial translation machineries make yeast a good model to quickly and efficiently evaluate the effect of variants in mtARS genes. Genetic screening of patients with a clinical suspicion of mitochondrial disorders through a customized gene panel of known disease-genes, including all genes encoding mtARSs, led to the identification of missense variants in WARS2, NARS2 and RARS2. Most of them were classified as Variant of Uncertain Significance. We exploited yeast models to assess the functional consequences of the variants found in these genes encoding mitochondrial tryptophanyl-tRNA, asparaginyl-tRNA, and arginyl-tRNA synthetases, respectively. Mitochondrial phenotypes such as oxidative growth, oxygen consumption rate, Cox2 steady-state level and mitochondrial protein synthesis were analyzed in yeast strains deleted in MSW1, SLM5, and MSR1 (the yeast orthologues of WARS2, NARS2 and RARS2, respectively), and expressing the wild type or the mutant alleles. Pathogenicity was confirmed for most variants, leading to their reclassification as Likely Pathogenic. Moreover, the beneficial effects observed after asparagine and arginine supplementation in the growth medium suggest them as a potential therapeutic approach.

Regucalcin Is a Potential Regulator in Human Cancer: Aiming to Expand into Cancer Therapy.

Regucalcin, a calcium-binding protein lacking the EF-hand motif, was initially discovered in 1978. Its name is indicative of its function in calcium signaling regulation. The rgn gene encodes for regucalcin and is situated on the X chromosome in both humans and vertebrates. Regucalcin regulates pivotal enzymes involved in signal transduction and has an inhibitory function, which includes protein kinases, protein phosphatases, cysteinyl protease, nitric oxide dynthetase, aminoacyl-transfer ribonucleic acid (tRNA) synthetase, and protein synthesis. This cytoplasmic protein is transported to the nucleus where it regulates deoxyribonucleic acid and RNA synthesis as well as gene expression. Overexpression of regucalcin inhibits proliferation in both normal and cancer cells in vitro, independent of apoptosis. During liver regeneration in vivo, endogenous regucalcin suppresses cell growth when overexpressed. Regucalcin mRNA and protein expressions are significantly downregulated in tumor tissues of patients with various types of cancers. Patients exhibiting upregulated regucalcin in tumor tissue have shown prolonged survival. The decrease of regucalcin expression is linked to the advancement of cancer. Overexpression of regucalcin carries the potential for preventing and treating carcinogenesis. Additionally, extracellular regucalcin has displayed control over various types of human cancer cells. Regucalcin may hold a prominent role as a regulatory factor in cancer development. Supplying the regucalcin gene could prove to be a valuable asset in cancer treatment. The therapeutic value of regucalcin suggests its potential significance in treating cancer patients. This review delves into the most recent research on the regulatory role of regucalcin in human cancer development, providing a novel approach for treatment.

Prevalence of anti-synthetase antibodies among systemic sclerosis patients

Backgroundwe aimed to estimate the prevalence of Amino acyl-transfer ribonucleic acid synthetase antibodies (Anti-ARS); myositis specific antibodies, among patients with systemic sclerosis (SSc), to evaluate the clinical associations of anti-ARS antibodies in SSc patients and to identify risk factors for development of interstitial lung disease (ILD) in SSc. MethodsA prospective study of 71 systemic sclerosis patients in our rheumatology clinic in Israel. Sera were tested for myositis antibodies. Data on patients clinical and serological manifestations and treatment were collected and compared according to anti-ARS antibodies and ILD. ResultsPrevalence of anti-ARS antibodies was 6% (4/71) with anti PL-7, anti- OJ and Jo-1 positivity. Anti Ro-52 was found in 27%, anti-PM/Scl 75, anti-PM/Scl 100 and anti-SRP in 6%, anti-Ku in 3%, anti-Mi-2 beta and anti-Mi-2 alfa in 4%, anti- NXP2 and anti-TIF1gamma in 1%. ILD complication was observed in 42% of patients and was associated with anti RNAP-III, anti Scl-70 and Anti-ARS antibodies. In multiple logistic regression, anti Scl-70 was associated with 6-fold higher risk for ILD. ConclusionAnti-ARS antibodies were observed in 6% of SSc patients. All of them had ILD. Due to the low prevalence of anti-ARS, this study could not describe clinical associations of anti-ARS antibodies in SSc patients.

High expression of VARS promotes the growth of multiple myeloma cells by causing imbalance in valine metabolism.

Multiple myeloma (MM) is a plasma cell malignancy occurring in middle and old age. MM is still an incurable disease due to its frequent recurrence and drug resistance. However, its pathogenesis is still unclear. Abnormal amino acid metabolism is one of the important characteristics of MM, and the important metabolic pathway of amino acids participates in protein synthesis as basic raw materials. Aminoacyl transfer ribonucleic acid synthetase (ARS) gene is a key regulatory gene in protein synthesis. This study aims to explore the molecular mechanism for ARS, a key factor of amino acid metabolism, in regulating amino acid metabolism in MM and affecting MM growth. The corresponding gene number was combined with the gene expression profile GSE5900 dataset and GSE2658 dataset in Gene Expression Omnibus (GEO) database to standardize the gene expression data of ARS. GSEA_4.2.0 software was used to analyze the difference of gene enrichment between healthy donors (HD) and MM patients in GEO database. GraphPad Prism 7 was used to draw heat maps and perform data analysis. Kaplan-Meier and Cox regression model were used to analyze the expression of ARS gene and the prognosis of MM patients, respectively. Bone marrow samples from 7 newly diagnosed MM patients were collected, CD138+ and CD138- cells were obtained by using CD138 antibody magnetic beads, and the expression of ARS in MM clinical samples was analyzed by real-time RT-PCR. Human B lymphocyte GM12878 cells and human MM cell lines ARP1, NCI-H929, OCI-MY5, U266, RPMI 8266, OPM-2, JJN-3, KMS11, MM1.s cells were selected as the study objects. The expression of ARS in MM cell lines was analyzed by real-time RT-PCR and Western blotting. Short hairpin RNA (shRNA) lentiviruses were used to construct gene knock-out plasmids (VARS-sh group). No-load plasmids (scramble group) and gene knock-out plasmids (VARS-sh group) were transfected into HEK 293T cells with for virus packaging, respectively. Stable expression cell lines were established by infecting ARP1 and OCI-MY5 cells, and the effects of knockout valyl-tRNA synthetase (VARS) gene on proliferation and apoptosis of MM cells were detected by cell counting and flow cytometry, respectively. GEO data were divided into a high expression group and a low expression group according to the expression of VARS. Bioinformatics analysis was performed to explore the downstream pathways affected by VARS. Gas chromatography time-of-flight mass spectrometry (GC-TOF/MS) and high performance liquid chromatography (HPLC) were used to detect the valine content in CD138+ cells and ARP1, OCI-MY5 cells and supernatant of knockdown VARS gene in bone marrow samples from patients, respectively. Gene enrichment analysis showed that tRNA processing related genes were significantly enriched in MM compared with HD (P<0.0001). Further screening of tRNA processing-pathway related subsets revealed that cytoplasmic aminoacyl tRNA synthetase family genes were significantly enriched in MM (P<0.0001). The results of gene expression heat map showed that the ARS family genes except alanyl-tRNA synthetase (AARS), arginyl-tRNA synthetase (RARS), seryl-tRNA synthetase (SARS) in GEO data were highly expressed in MM (all P<0.01). With the development of monoclonal gammopathy of undetermined significance (MGUS) to MM, the gene expression level was increased gradually. Kaplan-Meier univariate analysis of survival results showed that there were significant differences in the prognosis of MM patients in methionyl-tRNA synthetase (MARS), asparaginyl-tRNA synthetase (NARS) and VARS between the high expression group and the low expression group (all P<0.05). Cox regression model multivariate analysis showed that the high expression of VARS was associated with abnormal overall survival time of MM (HR=1.83, 95% CI 1.10 to 3.06, P=0.021). The high expression of NARS (HR=0.90, 95% CI 0.34 to 2.38) and MARS (HR=1.59, 95% CI 0.73 to 3.50) had no effect on the overall survival time of MM patients (both P>0.05). Real-time RT-PCR and Western blotting showed that VARS, MARS and NARS were highly expressed in CD138+ MM cells and MM cell lines of clinical patients (all P<0.05). Cell counting and flow cytometry results showed that the proliferation of MM cells by knockout VARS was significantly inhibited (P<0.01), the proportion of apoptosis was significantly increased (P<0.05). Bioinformatics analysis showed that in addition to several pathways including the cell cycle regulated by VARS, the valine, leucine and isoleucine catabolic pathways were upregulated. Non-targeted metabolomics data showed reduced valine content in CD138+ tumor cells in MM patients compared to HD (P<0.05). HPLC results showed that compared with the scramble group, the intracellular and medium supernatant content of ARP1 cells and the medium supernatant of OCI-MY5 in the VARS-shRNA group was increased (all P<0.05). MM patients with abnormal high expression of VARS have a poor prognosis. VARS promotes the malignant growth of MM cells by affecting the regulation of valine metabolism.

Aminoacyl transfer ribonucleic acid synthetase complex-interacting multifunctional protein 1 induces microglial activation and M1 polarization via the mitogen-activated protein kinase/nuclear factor-kappa B signaling pathway.

Activation of microglia, which is the primary immune cell of the central nervous system, plays an important role in neuroinflammation associated with several neuronal diseases. Aminoacyl tRNA synthetase (ARS) complex-interacting multifunctional protein 1 (AIMP1), a structural component of the multienzyme ARS complex, is secreted to trigger a pro-inflammatory function and has been associated with several inflammatory diseases. However, the effect of AIMP1 on microglial activation remains unknown. AIMP1 elevated the expression levels of activation-related cell surface markers and pro-inflammatory cytokines in primary and BV-2 microglial cells. In addition to the AIMP1-mediated increase in the expression levels of M1 markers [interleukin (IL)-6, tumor necrosis factor-α, and IL-1β], the expression levels of CD68, an M1 cell surface molecule, were also increased in AIMP-1-treated microglial cells, while those of CD206, an M2 cell surface molecule, were not, indicating that AIMP1 triggers the polarization of microglial cells into the M1 state but not the M2 state. AIMP1 treatment induced the phosphorylation of mitogen-activated protein kinases (MAPKs), while MAPK inhibitors suppressed the AIMP1-induced microglial cell activation. AIMP1 also induced the phosphorylation of the nuclear factor-kappa B (NF-κB) components and nuclear translocation of the NF-κB p65 subunit in microglial cells. Furthermore, c-Jun N-terminal kinase (JNK) and p38 inhibitors markedly suppressed the AIMP1-induced phosphorylation of NF-κB components as well as the nuclear translocation of NF-κB p65 subunit, suggesting the involvement of JNK and p38 as upstream regulators of NF-κB in AIMP1-induced microglial cell activation. The NF-κB inhibitor suppressed the AIMP1-induced M1 polarization of the microglial cells. Taken together, AIMP1 effectively induces M1 microglial activation via the JNK and p38/NF-κB-dependent pathways. These results suggest that AIMP1 released under stress conditions may be a pathological factor that induces neuroinflammation.

Identification of Similarities Between Skin Lesions in Patients With Antisynthetase Syndrome and Skin Lesions in Patients With Dermatomyositis by Highly Multiplexed Imaging Mass Cytometry.

Antisynthetase syndrome (ASyS) and dermatomyositis (DM) are autoimmune disorders that overlap clinically. Given the presence of DM-like skin lesions in ASyS patients, there is debate about whether ASyS is a distinct disease or a subclassification of DM. Recent studies identified differences in type I interferon (IFN) expression between ASyS and DM muscle and finger eruptions. This study was undertaken to elucidate similarities and differences in the pathogenesis of cutaneous disease in ASyS and DM at the single-cell level. Five ASyS patients and 7 DM patients were recruited from a prospectively collected database of well-characterized DM patients. ASyS patients were clinically confirmed as having ASyS according to the Connors et al criteria and the Solomon et al criteria and the presence of aminoacyl-transfer RNA synthetase antibodies. Immunophenotyping was conducted using immunofluorescence (IF) and imaging mass cytometry (IMC). IF staining for MxA and IFNβ expression revealed up-regulation of type I IFN in ASyS and DM samples compared to healthy control samples (P < 0.05). IMC showed similar numbers of macrophages, T cells, B cells, and dendritic cells in ASyS and DM samples, with no differences in counts (P > 0.05), but an increase in myeloid dendritic cell percentage in DM samples (P < 0.05). Key type I IFN, cytokine, and JAK/STAT pathways were similarly expressed in both ASyS and DM (P > 0.05). At the single-cell level, macrophages positive for phosphorylated stimulator of IFN genes in ASyS samples expressed increased levels of tumor necrosis factor, interluekin-17 (IL-17), and IFNβ (P < 0.001). IMC is a powerful tool that identifies a role for the type I IFN system in DM-like skin lesions in ASyS and DM with some differences at the cellular level, but overall significant overlap, supporting similar therapeutic decision making.

Acute digital ischemia: A rare presentation of antisynthetase syndrome.

Antisynthetase syndrome (ASS) is recognized as a subgroup of idiopathic inflammatory myopathies (IIMs). It is associated with autoantibodies directed against aminoacyl-transfer ribonucleic acid (tRNA) synthetase enzymes. We report the first case of anti-PL-7/anti-SSA 52kD ASS presenting as acute digital ischemia, an association not described previously. Occlusive vasculopathy is a rare but serious manifestation that can be seen at presentation in patients with ASS and may herald the onset of severe interstitial lung disease (ILD). Comprehensive evaluation should be performed to confirm the presence of subclinical myositis. Extensive myositis-specific antibody testing is strongly recommended even if initial screening autoimmune serologies are unrevealing.

The role of zinc as a stimulating factor in bone formation.

The role of zinc as a stimulatory factor on bone formation was reviewed. Bone growth retardation is a common finding in various conditions associated with zinc deficiency. The mechanism of zinc action on bone metabolism, however, has not been clarified so far. The oral administration of zinc sulfate (0.5-10 mg Zn/100 g body weight) to weanling rats can produce an increase in dry weight, alkaline phosphatase activity, calcium, collagen and deoxyribonucleic acid (DNA) contents in the femoral diaphysis, indicating that zinc has a stimulatory effect on bone formation and calcification. In tissue culture by using the calvaria from weanling rats, it has been demonstrated that zinc (10-6-10-4M) can stimulate bone protein synthesis, and that the zinc effect is based on activation of aminoacyl-transfer ribonucleic acid (tRNA) synthetase. The depletion of endogenous zinc in bone cells by treatment with dipicolinate, a chelator of zinc, can retard the synthesis of bone protein. Zinc is an essential factor in the systhesis of bone protein. Increasing age induces the deterioration of bone metabolism, and it decreases bone cellular zinc and protein systhesis. These decreases, however, can be restored by supplement of zinc. Thus, it has been introduced that zinc, an essential trace element, plays an important role in the stimulation of bone formation.

Régulation de la biosynthèse des aminoacyl tRNA synthétases des acides aminés branchés chez Bacillus cereus T

The regulation of the biosynthesis of isoleucyl-, valyl-, and leucyl-tRNA synthetases was studied with an isoleucine-valine requiring mutant of Bacillus cereus T. It was shown that valyl-tRNA synthetase regulation is under multivalent control involving both valine and isoleucine. The isoleucyl- and leucyl-tRNA synthetases are repressed by the respective cognate amino acid. When two amino acids were removed from the culture medium, derepression of the two corresponding aminoacyl tRNA synthetases was expected, but only one appears. With a threonine deaminase constitutive mutant, it was demonstrated that the derepression mecanism of the synthetase was correlated with the intracellular level of the ilv A gene product. These results are in good agreement with the model proposed by several authors. In this model, threonine deaminase, or some form of this enzyme, is involved in a positive control of the regulation of the branched-chain aminoacyltransfer ribonucleic acid synthetases.

Regulation of the biosynthesis of aminoacyl-transfer ribonucleic acid synthetases and of transfer ribonucleic acid in Escherichia coli. V. Mutants with increased levels of valyl-transfer ribonucleic acid synthetase

Spontaneous revertants of a temperature-sensitive Escherichia coli strain harboring a thermolabile valyl-transfer ribonucleic acid (tRNA) synthetase were selected for growth at 40 degrees C. Of these, a large number still contain the thermolabile valyl-tRNA synthetase. Three of these revertants contained an increased level of the thermolabile enzyme. The genetic locus, valX, responsible for the enzyme overproduction, is adjacent to the structural gene, valS, of valyl-tRNA synthetase. Determination (by radioimmunoassay) of the turnover rates of valyl-tRNA synthetase showed that the increased level of valyl-tRNA synthetase is due to new enzyme synthesis rather than decreased rates of protein degradation.

Regulation of the biosynthesis of aminoacyl-transfer ribonucleic acid synthetases and of transfer ribonucleic acid in Escherichia coli. VI. Mutants with increased levels of glutaminyl-transfer ribonucleic acid synthetase and of glutamine transfer ribonucleic acid

Spontaneous revertants of a temperature-sensitive Escherichia coli strain bearing a thermolabile glutaminyl-transfer ribonucleic acid (tRNA) synthetase have been selected for growth at 45 degrees C. Among 10 revertants still containing the thermolabile enzyme, 2 interesting strains were found. One strain has a fivefold elevated level of the thermolabile glutaminyl-tRNA synthetase; the genetic locus, glnR, responsible for this effect maps at min 24, far from glnS, the structural gene of the enzyme. In the other strain the levels of tRNA Gln and several other tRNAs are twice as high as in the parental strain; the locus responsible, glnU, maps at min 59.5 on the E. coli map.

Effects of Adenosine Triphosphate and Magnesium Chloride on Affinity Elution of Aminoacyl-Transfer Ribonucleic Acid Synthetases from Phosphocellulose with Transfer Ribonucleic Acids1

Aminoacyl-tRNA synthetases of bakers' yeast (Saccharomyces cerevisiae) were adsorbed to a phosphocellulose (P-cellulose) column, and those specific for tyrosine [EC 6.1.1.1], threonine [EC 6.1.1.3], valine [EC 6.1.1.9], and isoleucine [EC 6.1.1.5] were eluted with several specific tRNAs. Elutions of these synthetases were affected by ATP and/or MgCl2. The effects of ATP and MgCl2 differ with synthetases. Elutions of tyrosyl- and valyl-tRNA synthetases with their cognate tRNAs were more specific in the presence of MgCl2. Isoleucyl-tRNA synthetase was eluted with its cognate tRNA in the presence of both ATP and MgCl2. On the other hand, threonyl-tRNA synthetase was eluted in the absence of ATP and MgCl2 with unfractionated tRNA but not with some non-cognate tRNAs. This suggests that elution of threonyl-tRNA synthetase is highly specific. The present data on the effects of ATP or MgCl2 or both on this affinity elution will be useful for simple and rapid purification of the synthetases.

Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Cognate transfer ribonucleic acid as a ligand.

The use of tRNA affinity columns for the purification of aminoacyl-tRNA synthetases was investigated. A purification method for valyl-tRNA synthetase from Bacillus stearothermophilus is described that uses two affinity columns, one containing the pure cognate tRNA, and the other containing all tRNA species except the cognate tRNA. A method for the rapid preparation of the two columns was developed, which does not require prior isolation of cognate tRNA but makes use of the ability of the target synthetase to select its cognate tRNA. The usefulness of tRNA columns is compared with that of affinity columns derived from the aminoalkyladenylate reported in the preceding paper [Clarke & Knowles (1977) Biochem J. 167, 405-417].

Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Small organic ligands.

The usefulness of affinity chromatography for the purification of aminoacyl-tRNA synthetases was explored by using column ligands derived from the corresponding amino acid and aminoalkyladenylate, a non-labile analogue of the aminoacyladenylate reaction intermediate. Four modes of attachment of the aminoalkyladenylate to Sepharose were studied. The interaction between amino acid derivatives and the corresponding aminoacyl-tRNA synthetases is too weak to allow their use as ligands for affinity chromatography. Attachment of the aminoalkyladenylate via the alpha-nitrogen atom of the amino acid or via C-8 of the nucleotide abolishes synthetase binding, and immobilization via the oxidized ribose ring is only marginally useful. However, attachment of the aminoalkyladenylate to the matrix via N-6 of the nucleotide allows strong and specific synthetase binding, and the use of such columns permits the isolation of homogeneous synthetase from crude mixtures. The effect of non-specific adsorption and the utility of pre-columns and of specific substrate elution are investigated and discussed.

Detection and partial purification of rapidly sedimenting forms of aminoacyl-transfer ribonucleic acid synthetases from human placenta

The pattern of aggregation of aminoacyl-tRNA synthetases in extracts of human placenta is described. The sedimentation behavior of aminoacyl-tRNA synthetases in crude extracts of human placenta was analyzed by velocity gradient centrifugation. Varying proportions of seven aminoacyl-tRNA synthetase activities (arginyl-, glutaminyl-, glutamyl-, isoleucyl-, leucyl-, lysyl-, and methionyl-tRNA synthetases) sedimented as broad peaks at 17 to 21 S. Twelve other aminoacyl-tRNA synthetases sedimented at 5 to 9 S, a range more typical of individual aminoacyl-tRNA synthetases. The seven aminoacyl-tRNA synthetases which are aggregated in the placenta are the same enzymes which are most often reported to be aggregated in diverse mammalian species and tissues. All seven of the aggregated activities copurified as 16.2 to 18.8 S forms by ammonium sulfate precipitation, fractionation on alumina C γ and calcium phosphate gels, and chromatography on diethylaminoethyl (DEAE) cellulose. Before their gradient elution from DEAE-cellulose, the aggregated aminoacyl-tRNA synthetases could be stored with little loss of activity or change in sedimentation profile. Gradient elution of the aggregated activities from DEAE-cellulose columns resulted in accelerated loss of the activities during storage and in reduced sedimentation rates. Further reductions in sedimentation coefficients of aminoacyl-tRNA synthetases were observed after storage of fractions recovered from DEAE-cellulose columns.

Inhibition of aminoacyl-transfer ribonucleic acid synthetases and the regulation of amino acid biosynthetic enzymes in Neurospora crassa

Growth conditions that result in the accumulation of the tryptophan intermediate indoleglycerol phosphate or of the histidine intermediate imidazoleglycerol phosphate cause mycelia of Neurospora crassa to exhibit an immediate and sustained increase in the differential rate at which the biosynthetic enzymes of the tryptophan, histidine, and arginine pathways are synthesized. These accumulated intermediates are shown to be inhibitors of the activity of aminoacyltransfer ribonucleic acid (tRNA) synthetases, as judged by an in vitro esterification assay. The tryptophan intermediate is shown to inhibit the charging of tryptophan, and the histidine intermediate is shown to inhibit charging of histidine. The inhibitions noted are consistent with the finding that the level of charged tRNATrp is decreased significantly in cells that have accumulated indoleglycerol phosphate and that of tRNAHis is decreased significantly in cells that have accumulated imidazoleglycerol phosphate. These results are interpreted as support for the involvement of aminoacyl-tRNA species in mediating cross-pathway regulation of the tryptophan, histidine, and arginine biosynthetic pathways as proposed in Lester's polyrepressor hypothesis (G. Lester, 1971). the correlations noted lead to the conclusion that Neurospora utilizes regulatory mechanisms that have the ability to react to changes in the level of charging of tRNA species.

Transient rates of synthesis of five amionacyl-transfer ribonucleic acid synthetases during a shift-up of Escherichia coli

The steady-state levels of a number of aminoacyl-transfer ribonucleic acid synthetases are known to be positively correlated with growth rate in Escherichia coli. To describe the regulation of these enzymes during a nutritional shift-up, use was made of the recent identification of polypeptide chains of several synthetases in whole cell lysates resolved by the O'Farrell two-dimensional gel system. A culture growing in acetate minimal medium was shifted to glucose-rich medium and pulse labeled with [3H]leucine and [3H]isoleucine for 30- or 6-s intervals during the 20 min after the shift. After mixing with a uniformly [35S]sulfate-labeled reference culture, the samples were subjected to two-dimensional gel electrophoresis. The 3H/35S ratio in the resolved synthetase polypeptides provided an accurate estimation of their transient rates of synthesis. Five aminoacyl-transfer ribonucleic acid synthetases (those for argnine, glycine, isoleucine, phenylalanine, and valine) exhibited an increase in formation within 30 to 90 s after the shift-up. The magnitude of the increases corresponded to the final steady-state values and were reached within 2 to 3 min. The addition to rifampin revealed that the increase in the differential rate of valyl-transfer ribonucleic acid synthetase formation was the result of increased messenger ribonucleic acid transcription and not of a relaxation of some translation restriction.

Chemical measurement of steady-state levels of ten aminoacyl-transfer ribonucleic acid synthetases in Escherichia coli

Polypeptide chains of 10 aminoacyl-transfer ribonucleic acid synthetases (those for arginine, glutamine, glutamic acid, glycine, isoleucine, leucine, lysine, phenylalanine, threonine, and valine) have been identified in lysates of Escherichia coli resolved by the O'Farrell two-dimensional gel system. By labeling cells uniformly with [14C]glucose and by measuring the total amounts of these polypeptides by their radioactivity, estimations of the steady-state, molecular amounts of these enzymes were made and compared to the number of ribosomes and elongation factors in these cells. Portions of a reference culture grown on glucose and labeled with [14C]leucine or [35S]sulfate were mixed with four cultures grown in widely different media containing [3H]leucine or [3H]leucine plus [3H]isoleucine. From the isotope ratios of the total protein and of the spots containing the synthetase chains, the chemical amount of each synthetase relative to that of the reference culture was determined. The results, where comparable, show reasonable agreement with enzyme activity measurements. In general, these synthetases each exhibit a positive correlation with growth rate in unrestricted media, indicating a strong tendency for the levels of transfer ribonucleic acid, synthetases, elongation factors, and ribosomes to remain approximately, though not exactly, in balance at different growth rates.

Growth rate modulation of four aminoacyl-transfer ribonucleic acid synthetases in enteric bacteria

The specific activities of arginyl- glutamyl- seryl-, and valyl-transfer ribonucleic acid (tRNA) synthetases were measured in the wild-type and mutant strains of Salmonella typhimurium LT2 and Escherichia coli B/r. In media restricted only by carbon and energy source availability, the specific activities of all four enzymes were proportional to the growth rate, with the exception of seryl-tRNA synthetase in S. typhimurium, which remained essentially constant. Structural gene densities were calculated for these four enzymes and were found not to account for the variation of specific activity with growth rate.

Three photo-cross-linked complexes of yeast phenylalanine specific transfer ribonucleic acid with aminoacyl transfer ribonucleic acid synthetases.

Yeast tRNA-Phe has been cross-linked photochemically to three aminoacyl-tRNA synthetases, yeast phenylalanyl-tRNA synthetase, Escherichia coli isoleucyl-tRNA synthetase, and E. coli valyl-tRNA synthetase. The two non-cognate enzymes are known to interact with tRNA-Phe. In each complex, three regions on the tRNA are found to cross-link. Two of these are common to all of the complexes, while the third is unique to each. Thus, the cognate and non-cognate complexes bear considerable similarity to each other in the way in which the respective enzyme orients on tRNA-Phe, a result which was also established for the complexes of E. coli tRNA-Ile (BUDZIK, G.P., LAM, S.M., SCHOEMAKER, H.J.P., and SCHIMMEL, P.R. (1975) J. Biol. Chem. 250, 4433-4439). The common regions include a piece extending from the 5'-side of the acceptor stem to the beginning of the dihydrouridine helix, and a segment running from the 3' side of the extra loop into the TpsiC helix. These two regions overlap with and include some of the homologous bases found in eight tRNAs aminoacylated by yeast phenylalanyl-tRNA synthetase (ROE, B., SIROVER, M., and DUDOCK, B. (1973) Biochemistry 12, 4146-4153). Although well separated in the primary and secondary structure, these two segments are in close proximity in the crystallographic tertiary structure. In two of the complexes, the third cross-linked fragment is near to the two common ones. The picture which emerges is that the enzymes all interact with the general area in which the two helical branches of the L-shaped tertiary structure fuse together, with additional interactions on other parts of the tRNAas well.