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Results for “Alanine-tRNA Ligase”

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At least 19 recordsLinked to original sources
PubMed2005

Treatment of antisynthetase-associated interstitial lung disease with tacrolimus.

OBJECTIVE: To assess the efficacy of tacrolimus in patients with anti-aminoacyl-transfer RNA synthetase (anti-aaRS)-associated interstitial lung disease (ILD) and idiopathic inflammatory myopathy (IIM). METHODS: Ninety-eight patients with anti-aaRS autoantibodies were identified in our IIM cohort of 536 patients. The medical records of 15 patients with anti-aaRS-associated ILD treated with tacrolimus between 1992 and 2003 were retrospectively reviewed. Pulmonary parameters of response included forced vital capacity, forced expiratory volume in 1 second, and diffusing capacity for carbon monoxide. Manual muscle testing results, serum creatine kinase (CK) levels, and the daily corticosteroid dosage were used to assess improvement in myositis. Random coefficient modeling considering polynomials of time was used to assess the clinical response to tacrolimus. RESULTS: All patients, except for 1, who had pure ILD, had definite or probable IIM. Two patients received tacrolimus for fewer than 3 months, and their data were not analyzed. For the remaining 13 patients, the mean age at onset of ILD was 46.9 years, and the mean duration of pulmonary disease was 14.7 months. Twelve patients had anti-histidyl-transfer RNA synthetase autoantibody (anti-Jo-1) and 1 had anti-alanyl-transfer RNA synthetase autoantibody (anti-PL-12). Patients received tacrolimus for an average of 51.2 months. A significant improvement was observed in all pulmonary parameters measured. The serum CK level declined significantly, and 10 patients had either an improvement in muscle strength or maintained normal muscle strength. A statistically significant reduction in the corticosteroid dosage was also observed. CONCLUSION: Tacrolimus is a well-tolerated and effective therapy for managing refractory ILD and myositis in anti-aaRS-positive patients.

Adult↗
PubMed2006

Unusually high frequency of autoantibodies to PL-7 associated with milder muscle disease in Japanese patients with polymyositis/dermatomyositis.

OBJECTIVE: Autoantibodies to aminoacyl transfer RNA synthetases, such as histidyl (Jo-1), threonyl (PL-7), alanyl (PL-12), glycyl (EJ), and isoleucyl (OJ), are closely associated with a subset of patients with polymyositis/dermatomyositis (PM/DM) complicated by interstitial lung disease (ILD). Anti-Jo-1 is by far the most common, found in 15-25% of patients with PM/DM, whereas the other types are found in only approximately 3% of these patients. In this study, the clinical associations of these autoantibodies in Japanese patients with PM/DM were investigated. METHODS: The diagnoses of PM/DM and amyopathic DM (ADM) were based on the Bohan and Peter criteria and Sontheimer's definition, respectively. Sera from 36 Japanese patients with PM/DM (13 with PM, 20 with DM, 3 with ADM) were screened by immunoprecipitation and by enzyme-linked immunosorbent assay (for Jo-1). Clinical and laboratory data were collected. RESULTS: The frequencies of autoantibodies to Jo-1 (22%) and to EJ, OJ, and PL-12 (3-6%) were similar to those found in previous studies, including studies of Japanese subjects. However, anti-PL-7 was found in 17% of patients, in contrast to a frequency of 1-4% in previous studies (P < 0.02-0.0002). The 6 anti-PL-7-positive patients were not related, and no skewing in year or month of disease development, place of residence or work, or occupation was found. All patients had ILD, consistent with the clinical features of antisynthetase-positive patients. The patients with anti-PL-7 had lower serum muscle enzyme levels and milder muscle weakness (P < 0.05) compared with anti-Jo-1-positive patients. CONCLUSION: Anti-PL-7 was found at an unusually high frequency in this group of Japanese patients with myositis. Although anti-PL-7, similar to anti-Jo-1, is associated with PM/DM with ILD, muscle involvement in the patients with anti-PL-7 appeared to be milder than that in the anti-Jo-1 subset.

Adult↗
PubMed1992

Penicillin binding protein 2 is dispensable in Escherichia coli when ppGpp synthesis is induced.

Mecillinam, a beta-lactam antibiotic which specifically inactivates penicillin binding protein 2 (PBP2) in Escherichia coli, prevents lateral cell wall elongation, inducing spherical morphology and cell death. Two mecillinam resistant mutants, lov-1 and lovB, both able to dispense entirely with PBP2, are shown here to be affected in the aminoacyl-tRNA synthetase genes argS and alaS, respectively. Although the argS and alaS mutants grow slowly, we show that there is no correlation between mecillinam resistance and either growth rate or translation speed. A role of the ribosomes in mecillinam sensitivity, suggested by our earlier report that the lov-1 mutation is suppressed by certain rpsL(StrR) alleles affecting ribosomal protein S12, is supported by the present observation that a pseudo-streptomycin dependent mutant is mecillinam resistant in the presence of streptomycin. The argS and alaS mutants have high pools of the nucleotide ppGpp (effector of the stringent response) and the mecillinam resistance of both mutations is suppressed by a relA mutation, inactivating the ribosome-associated ppGpp synthetase and preventing ppGpp synthesis in response to aminoacyl-tRNA starvation. Furthermore, a ptacrelA' multicopy plasmid makes a wild type strain mecillinam resistant. The effect of ppGpp is probably mediated by RNA polymerase, since sublethal doses of the polymerase inhibitor rifampicin suppress mecillinam resistance in argS, alaS and ptacrelA'-bearing strains. We conclude that ppGpp regulates the transcription of a gene whose product is involved in mecillinam sensitivity, possibly as part of a chain of interacting elements which coordinate ribosomal activity with that of the PBPs.

Alanine-tRNA Ligase↗
PubMed1995

Cell growth inhibition by sequence-specific RNA minihelices.

RNA minihelices which reconstruct the 12 base pair acceptor-T psi C domains of transfer RNAs interact with their cognate tRNA synthetases. These substrates lack the anticodons of the genetic code and, therefore, cannot participate in steps of protein synthesis subsequent to aminoacylation. We report here that expression in Escherichia coli of either of two minihelices, each specific for a different amino acid, inhibited cell growth. Inhibition appears to be due to direct competition between the minihelix and its related tRNA for binding to their common synthetase. This competition, in turn, sharply lowers the pool of the specific charged tRNA for protein synthesis. Inhibition is relieved by single nucleotide changes which disrupt the minihelix-synthetase interaction. The results suggest that sequence-specific RNA minihelix substrates bind to cognate synthetases in vivo and can, in principle, act as cell growth regulators. Naturally occurring non-tRNA substrates for aminoacylation may serve a similar purpose.

Alanine-tRNA Ligase↗
PubMed1993

Amino acid substitutions at position 73 in motif 2 of Escherichia coli alanyl-tRNA synthetase.

Lysine73, located in the adenylate synthesis domain of Escherichia coli alanyl-tRNA synthetase (AlaRS), was previously indicated to be an important residue for the interaction of this enzyme with the acceptor stem of its cognate tRNA (tRNA(Ala)). Replacement of this residue with glutamine produced a reduction in the catalytic efficiency of AlaRS in the aminoacylation assay, primarily through an increase in the apparent KM for tRNA(Ala) [Hill, K., and Schimmel, P. (1989) Biochemistry 28, 2577-2586]. Studies on the role of residue 73 in the interaction of AlaRS with its substrates have now been extended using the additional substitutions of asparagine, alanine, and glutamate. Analysis of each substituted enzyme in the ATP-PPi exchange and aminoacylation reactions reveals kinetic characteristics similar to those obtained with the glutamine substitution, except that the glutamate substitution causes a fivefold decrease in the affinity for alanine. These data verify that the positive charge on lysine 73, rather than its hydrophilic side chain, is of importance in the binding of the cognate tRNA, but do not support an ionic interaction of this residue with the RNA phosphate backbone. The collective data support the prediction that lysine73 is in motif 2 of AlaRS [Cusack, S., Hartlein, M., and Leberman, R. (1991) Nucleic Acids Res. 19, 3489-3498], but question the predicted alignment of this motif with other enzymes in its class.

Alanine-tRNA Ligase↗
PubMed1996

Further characterization of Escherichia coli alanyl-tRNA synthetase.

Selected physical and thermodynamic parameters for Escherichia coli alanyl-tRNA synthetase (AlaRS) have been determined primarily to assess the quaternary structure of this enzyme. The extinction coefficient (epsilon) at 280 nm was determined experimentally to be 0.71 ml mg-1 cm-1, and the partial specific volume (nu) was calculated from the amino acid composition to be 0.73 ml g-1. From viscosity experiments the intrinsic viscosity (eta) of AlaRS was extrapolated to be 3.4 ml g-1 and the degree of hydration (delta 1) estimated to be 0.67 gH2O g(-1)(AlaRS). Laser light-scattering studies indicated some heterogeneity; a radius of 6.3 nm was calculated for the major fraction with a diffusion coefficient (D20,W) of 3.89 x 10(-7) cm2 s-1. In 50 mM Hepes, pH 7.5, 20 mM KCl, 2 mM 2-mercaptoethanol and at a protein concentration of 4.2 mg ml-1 the sedimentation coefficient (S20,W) was 6.36 S; this value increased slightly when the protein concentration was decreased. The combination of S20,W and D20,W under these conditions yielded a molecular weight of approximately 186,000 Da, corresponding to a dimer. The S20,W was virtually independent of temperature in the range of 10-37 degrees C, while an Arrhenius plot of aminoacylation activity was biphasic. The isoelectric point was determined experimentally to be 4.9. Sedimentation equilibrium data were best fit to a decamer association complex in which dimeric AlaRS is the predominant species at 25 degrees C.

Alanine-tRNA Ligase↗
PubMed1999

Characterization of zinc-depleted alanyl-tRNA synthetase from Escherichia coli: role of zinc.

To evaluate the role of zinc in Escherichia coli alanyl-tRNA synthetase, hydrodynamic measurements and circular dichroism spectra were obtained for the zinc-depleted protein and compared with those of the native enzyme. At a protein concentration of 5 mg ml(-1), pH 7.5, the sedimentation coefficient (s(20,w)) was 6.3 S and was virtually independent of temperature between 10 and 37 degrees C, similar to the results reported for the native form. However, the s(20,w) now decreased significantly as the concentration increased, indicative of a possible change in conformation. The s(20,w) value did not appear to change as the pH was increased to 9.5. In standard buffer with 3.3 M added urea, a single peak with a s(20,w) of 3.6 S was obtained and with 6.6 M added urea, a peak with a s(20,w) of 2.7 S was seen. Added Gd-HCl (6 M) gave a single peak with s(20,w) of 2. 0 S. Like the native form, laser light scattering studies indicated some heterogeneity and a radius of 6.4 nm which was virtually independent of concentration and temperature in the range of 10-37 degrees C. At 25 degrees C, a diffusion coefficient (D(20,w)) of 3.3 x 10(-7) cm(2) s(-1) was obtained. The combination of s(0)(20,w) and D(20,w) yielded a molecular mass of approximately 179 kDa, which is slightly less than that reported for the native dimeric form (186 kDa). The intrinsic viscosity at 25 degrees C was extrapolated to 5. 3 ml g(-1), a value significantly higher than that reported for the native form, which increased with temperature. These results indicate some conformational and flexibility changes from the native to the zinc-depleted form, which may explain differences in activity. Furthermore, urea denaturation experiments demonstrate the role of zinc in stabilization of AlaRS structure.

Alanine-tRNA Ligase↗
PubMed1993

A continuous spectrophotometric assay for the aminoacylation of transfer RNA by alanyl-transfer RNA synthetase.

A continuous spectrophotometric assay is described for the aminoacylation reaction catalyzed by Escherichia coli alanyl-transfer RNA synthetase. The assay is based on coupling the alanyl-tRNA synthetase-dependent formation of AMP to the lactate dehydrogenase oxidation of NADH. Oxidation of NADH, as monitored at 340 nm, is shown to be stoichiometric with the formation of alanyl-tRNA(Ala). This assay will facilitate the rapid accumulation and analysis of kinetic data for alanyl-tRNA synthetase and should be applicable to aminoacyl-tRNA synthetases in general.

Acylation↗
PubMed1994

Cooperative binding of zinc to an aminoacyl-tRNA synthetase.

Zinc binds to tetrameric alanyl-tRNA synthetase from Escherichia coli with a stoichiometry of one g-atom of zinc per enzyme subunit. The nature of this metal-protein interaction is investigated here through a series of equilibrium dialysis and intrinsic fluorescence experiments. The dialysis data show that zinc binds to this synthetase in a cooperative manner, with half-maximal zinc binding at 0.97 microM free zinc and a Hill coefficient of 1.9. The cooperative feature is also observed in the zinc-induced quenching of the protein intrinsic fluorescence, indicating that zinc binding induces a conformational change. This is the first report of cooperative binding of zinc to an aminoacyl-tRNA synthetase, and the data provide a rationale for the oligomeric structure of the synthetase specific for alanine.

Alanine-tRNA Ligase↗
PubMed1993

Mapping of the zinc binding domain of Escherichia coli methionyl-tRNA synthetase.

Cys/His motifs, found in several nucleic acid binding proteins, generally correspond to sites for the binding of metal atoms. Such a motif, comprising four Cys residues, occurs in the subunits of Escherichia coli methionyl-tRNA synthetase, a dimeric enzyme known to bind two zinc atoms. In this study, each of the four cysteines in the cysteine cluster (region 145 to 161) of E. coli methionyl-tRNA synthetase were successively changed into an alanine. Either substitution is sufficient to destabilize the tight binding of the zinc ion. Moreover, a peptide having a sequence corresponding to that of the 138 to 163 region of methionyl-tRNA synthetase has been prepared. It strongly binds one zinc atom, even in the presence of ethylene diamine tetraacetate. These data establish that, in methionyl-tRNA synthetase, the Cys motif of region 145 to 161 is actually the binding site for zinc. In addition, the mutation of each cysteine modifies the parameters of the methionine activation reaction, and appears to change the structure of the enzyme, as probed by an increased sensitivity of the mutant enzymes to trypsin attack. The possible role of the zinc atom and of its chelating residues in the folding of the active centre of methionyl-tRNA synthetase is discussed.

Adenosine Triphosphate↗
PubMed1999

The reliability of in vivo structure-function analysis of tRNA aminoacylation.

The G.U wobble base-pair in the acceptor helix of Escherichia coli tRNAAlais critical for aminoacylation by the alanine synthetase. Previous work by several groups probed the mechanism of enzyme recognition of G.U by a structure-function analysis of mutant tRNAs using either a cell assay (amber suppressor tRNA) or a test tube assay (phage T7 tRNA substrate and purified enzyme). However, the aminoacylation capacity of particular mutant tRNAs was about 10(4)-fold higher in the cell assay. This led us to scrutinize the cell assay to determine if any parameter exaggerates the extent of aminoacylation in mutants forming substantial amounts of alanyl-tRNAAla. In doing so, we have refined and developed experimental designs to analyze tRNA function. We examined the level of aminoacylation of amber suppressor tRNAAlawith respect to the method of isolating aminoacyl-tRNA, the rate of cell growth, the cellular levels of alanine synthetase and elongation factor TU (EF-Tu), the amount of tRNA and the characteristics of EF-Tu binding. Within the precision of our measurements, none of these parameters varied in a way that could significantly amplify cellular alanyl-tRNAAla. A key observation is that the extent of aminoacylation of tRNAAlawas independent of tRNAAlaconcentration over a 75-fold range. Therefore, the cellular assay of tRNAAlareflects the substrate quality of the molecule for formation of alanyl-tRNAAla. These experiments support the authenticity of the cellular assay and imply that a condition or factor present in the cell assay may be absent in the test tube assay.

Acylation↗
PubMed2001

Simultaneous and functional binding of SmpB and EF-Tu-TP to the alanyl acceptor arm of tmRNA.

Transfer-messenger RNA (tmRNA) mimics functions of aminoacyl-tRNA and mRNA, subsequently, when rescuing stalled ribosomes on a 3' truncated mRNA without stop codon in bacteria. In addition, this mechanism marks prematurely terminated proteins by a C-terminal peptide tag as a signal for degradation by specific cellular proteases. For Escherichia coli, previous studies on initial steps of this "trans-translation" mechanism revealed that tmRNA alanylation by Ala-tRNA synthetase and binding of Ala-tmRNA by EF-Tu-GTP for subsequent delivery to stalled ribosomes are inefficient when compared to analogous reactions with canonical tRNA(Ala). In other studies, protein SmpB and ribosomal protein S1 appeared to bind directly to tmRNA and to be indispensable for trans-translation. Here, we have searched for additional and synergistic effects of the latter two on tmRNA alanylation and its subsequent binding to EF-Tu-GTP. Kinetic analysis of functioning combined with band-shift experiments and structural probing demonstrate, that tmRNA may indeed form a multimeric complex with SmpB, S1 and EF-Tu-GTP, which leads to a considerably enhanced efficiency of the initial steps of trans-translation. Whereas S1 binds to the mRNA region of tmRNA, we have found that SmpB and EF-Tu both interact with its acceptor arm region. Interaction with SmpB and EF-Tu was also observed at the acceptor arm of Ala-tRNA(Ala), but there the alanylation efficiency was inhibited rather than stimulated by SmpB. Therefore, SmpB may function as an essential modulator of the tRNA-like acceptor arm of tmRNA during its successive steps in trans-translation.

Acylation↗
PubMed1994

Characterization of some major identity elements in plant alanine and phenylalanine transfer RNAs.

Alanine and phenylalanine tRNA sequences were amplified by PCR from Arabidopsis thaliana nuclear DNA using degenerate oligonucleotides which introduced specific mutations into the acceptor stem. The aminoacylation of T7 RNA polymerase transcripts of these sequences was investigated in vitro using partially purified bean alanyl- or phenylalanyl-tRNA synthetase. In parallel, the in vivo activity of amber suppressor derivatives of these tRNAs was investigated in transient expression assays in tobacco protoplasts using a beta-glucuronidase (GUS) reporter gene containing a premature amber stop codon. The results show that mutation of the G3:U70 base pair to G3:C70 blocks aminoacylation of plant alanine tRNA, whilst conversion of the G3:C70 pair normally found in plant tRNA(Phe) to G3:U70 enables the mutated tRNA(Phe) to be a good substrate for alanyl-tRNA synthetase and impairs its aminoacylation with phenylalanine. In addition, the amber suppressor derivative of wild-type tRNA(Phe) showed very little suppressor activity in vivo, and was poorly aminoacylated with phenylalanine in vitro, suggesting that the anticodon is a major identity determinant for tRNA(Phe) in plant cells.

Alanine-tRNA Ligase↗