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Results for “Amino Acyl-tRNA Synthetases”

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At least 19 recordsLinked to original sources
PubMed1976

Effect of excision of the Y-base on the interaction of tRNAPhe (yeast) with phenylalanyl-tRNA synthetase (yeast).

The interaction between tRNAPhe (yeast), from which the Y-base has been removed by acid treatment, and phenylalanyl-tRNA synthetase (yeast) has been investigated by fluorescence competition titrations and sedimentation velocity runs. The binding parameters are given under various ionic conditions. The tRNAPhe-Y still can occupy the specific binding sites on the enzyme. Compared to unmodified tRNAPhe, the binding constant is lowered by more than one order of magnitude. It can be concluded that the Y-base is not necessary for specific recognition of tRNAPhe by the cognate synthetase, it rather may represent a point of attachment for the synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1979

Aminoacyl-tRNA synthetase mutants degrade protein at a normal rate.

The stability of both rapidly and slowly degraded proteins in wild type CHO cells is similar to that in three ts aminoacyl-tRNA synthetase mutants at both permissive and non-permissive temperatures, although the degree of tRNA charging in the synthetase mutants differs considerably with temperature. These results indicate that the altered rate of protein breakdown seen under a variety of physiological conditions in eukaryotic systems is not mediated by uncharged tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed1975

Aminoacyl transfer from an adenylate anhydride to polyribonucleotides.

Imidazole catalysis of phenylalanyl transfer from phenylalanine adenylate anhydride to the hydroxyl groups of homopolyribonucleotides was investigated as a chemical model of the biochemical aminoacylation of tRNA. Imidazole catalyzed transfer of phenylalanine to poly(U) increases from pH 6.5 to 7.7 and decreases above pH 7.7. At pH 7.7 approximately 10% of the phenylalanyl residues are transferred to poly(U). At pH 7.1, transfer to poly(U) was five times as great as to poly(A) and transfer to a poly(A) poly(U) double helix was negligible. At pH 7.1 approximately 45 mole percent linkages to poly(U) were monomeric phenylalanine; the remainder of the linkages were peptides of phenylalanine. The number of linkages and their lability to base and neutral hydroxylamine indicates that phenylalanine and its peptides are attached as esters to the 2' hydroxyl groups throughout poly(U) and the 2' (3') hydroxyl groups at the terminus of poly(U). These results do model the contemporary process of aminoacyl transfer to tRNA and continue to suggest that a histidine residue is in the active site of aminoacyl-tRNA-synthetases.

Adenosine Monophosphate↗
PubMed1976

Distinct steps in the specific binding of tRNA to aminoacyl-tRNA synthetase. Temperature-jump studies on the serine-specific system from yeast and the tyrosine-specific system from Escherichia coli.

The kinetics of the interaction of tRNASer and seryl-tRNA synthetase from yeast as well as of tRNATyr and tyrosyl-tRNA synthetase from Escherichia coli have been investigated by temperature-jump experiments. It could be shown that complex formation proceeds in two distinct steps. This was demonstrated for both the first and the second binding site. The two-step mechanism was deduced from the characteristic concentration dependence of the relaxation times. Seryl-tRNA synthetase recombines with the first tRNA to form an intermediate complex (kI12, kI21), which is transformed in a fast reaction to the final 1:1 complex (kI23, kI32). At pH 7.2 with 0.1 M KCl the rate constants are: kI12 = 2.7 X 10(8) M-1 S-1; kI23, kI32). At pH 7.2 with 0.1 M KCl the rate constants are: kI12 = 2.7 x 10(8) M-1 S-1; kI21 = 220 S-1; kI23 = 760 S-1; kI32 = 330 S-1. The 1:1 complex can bind a second tRNA. At pH 7.2 without added salt the rate constants are: KII2 = 0.9 X 10(8) M-1 S-1; kII21 = 270 S-1; kII23 = 120 S-1; kII32 = 1250 S-1. The tyrosine-specific system behaves very similarly to the serine-specific system. Data are given for pH 7.2 (pH 6.0) for the binding of the second tRNA: kII12 = 1 X 10(8) (2.5 X 10(8)) M-1 S-1; kII21 = 470 (170) S-1; kII23 = 150 (530) S-1; kII32 = 1540 (720) S-1. The kinetic results are discussed in terms of their relevance to the recognition process and their relation to the anticooperative binding behaviour of tRNA to synthetase.

Amino Acyl-tRNA Synthetases↗
PubMedDate not supplied

[Activation of L-amino acids by aminoacyl-tRNA-synthetases from yeast Candida utilis IBPM-405].

The procedure for isolating aminoacyl-tRNA-synthetases from yeast Candida utilis IBPM-405 was developed. The rate of activation of L-amino acids in the formation of hydroxamates was different. Aspartic acid, asparagine, glutamic acid, tryptophane, phenyl alanine and methionine underwent the highest activation. The activation of alanine, arginine, hydroxyproline, serine and isoleucine was insignificant. Using aspartic acid, it was shown that the hydroxamate formation was ATP-stimulated and that the amount of hydroxamate increased with a rise of the protein concentration in the mixture to 9-10 mg/ml. The hydroxamate formation was inhibited by p-chloromercury-benzoate and heavy metal ions. Yeast aminoacyl-tRNA-synthetases showed L-aspartic and L-glutamic activities that were independent from Mg++ ions and ATP.

Adenosine Triphosphate↗
PubMed1975

A reinvestigation of the sites of transcription and translation of Euglena chloroplastic phenylalanyl-tRNA synthetase.

An attempt was made to determine the sites of chloroplast phenylalanyl-tRNA synthetase transcription and translation. Inhibitors of bacterial RNA and protein synthesis were added to logarithmic and stationary phase cultures of Euglena gracilis wild-type B. Logarithmic phase cultures were sensitive to both types of inhibitors. In stationary phase cultures plastid synthetase was reduced by RNA but not by protein synthesis inhibitors. The effect of the antibiotics on the mitochondrial enzyme was also noted. Several possible explanations of these resuults are discussed.

Amino Acyl-tRNA Synthetases↗
PubMed1975

Complete purification and studies on the structural and kinetic properties of two forms of yeast valyl-tRNA synthetase.

Two forms of baker's yease valyl-tRNA synthetase have been purified to apparent homogeneity by classical methods. It was demonstrated that one of the two forms of the enzyme originates from the other by proteolysis, the respective amounts of each form depending on the physiological state of the yeast. The species mainly isolated from exponential growing yeast cells is a monomer of 130,000 daltons molecular weight. In stationary phase cells or in commercial yeast the major species is a degraded monomer of 120,000 daltons molecular weight ; however when the purification is carried out in the presence of phenylmethyl-sulphonyl fluoride, or diisopropylfluorophosphate large amounts of the not - degreded monomer can be obtained. Of great practical usefulness is the fact that large amounts of the native enzyme can be obtained pure after only two chromatographic steps on DEAE-cellulose and hydroxylapatite. The kinetic constants for valine, ATP and tRNAVal were determined, as well as the optimum aminoacylation conditions. It was found that the specific activity of the nondegraded valyl-tRNA synthetase is higher than that of the proteolysed enzyme for the aminoacylation reaction. On the contrary, both forms have the same ATP-pyroposphate exchange activity. The amino acids composition of the native enzyme was established. The tryptic fingerprints of the two valyl-tRNA synthetases were studied. Essentially similar maps were obtained. The number of the spots in the fingerprints indicates that the enzymes contain a high proportion of repeated sequences.

Adenosine Triphosphate↗
PubMed2025

Genetic Incorporation of a Thioxanthone-Containing Amino Acid for the Design of Artificial Photoenzymes.

Genetically encodable photosensitizers allow the design of artificial photoenzymes to expand the scope of abiological reactions. Herein, we report the genetic incorporation of a thioxanthone-containing amino acid into a protein scaffold via an engineered pyrrolysyl-tRNA/pyrrolysyl-tRNA synthetase pair. The designer enzyme was engineered to catalyze a dearomative [2+2] cycloaddition reaction in high yields (up to>99 % yield) with excellent enantioselectivity (up to 98 : 2 e.r.). This work provides a robust and facile method for photoenzyme design and lays the foundation for the development of further photoenzymatic reactions.

Xanthones↗
PubMed1976

Histidyl transfer ribonucleic acid synthetase from Salmonella typhimurium. Interaction with substrates and ATP analogues.

Structural requirements for substrate binding to histidyl-tRNA synthetase from Salmonella typhimurium have been investigated using ATP analogues. Ki values and the relative binding affinity of the enzyme for these analogues have been determined in the tRNA aminoacylation reaction. The enzyme is highly specific for ATP: no binding was found for GTP, CTP, TTP and UTP. dATP is a very poor substrate for acylation of tRNA, with a Km 40-fold higher than that of ATP. Binding of adenosine 5'-triphosphate requires interactions of the amino group of adenosine and the sugar moiety; the 2' and the 5' positions of the ribose appear to be essential for recognition; the phosphate groups enhance the binding. AMP is a noncompetitive inhibitor with ATP. The interaction of histidyl-tRNA synthetase, a dimeric enzyme, with histidine and ATP was examined by fluorescence measurements at equilibrium and by equilibrium dialysis. Binding with L-histidine is significantly tighter at pH 6 than at pH 7, while the ATP binding is independent of pH. The stoichiometry was measured at pH 6 than at pH 7, while the ATP binding is independent of pH. The stoichiometry was measured at pH 7.5 by equilibrium dialysis and is 1 mol ATP/mol enzyme and, variably, close to 2 or 1 mol histidine/mol enzyme.

Adenosine Triphosphate↗
PubMed2025

Loss-of-Function CARS1 Variants in a Patient With Microcephaly, Developmental Delay, and a Brittle Hair Phenotype.

BACKGROUND: Mutations in cysteinyl-tRNA synthetase (CARS1) have been implicated in a multisystem disease including microcephaly, developmental delay, and brittle hair and nail phenotypes. METHODS: Here, we present a patient with hepatopathy, hypothyroidism, short stature, developmental delay, microcephaly, muscular hypotonia, brittle hair, and ataxia. The patient underwent exome sequencing to identify potentially pathogenic genetic variants. In addition, identified variants were assessed using yeast complementation assays to determine functional consequences. RESULTS: Exome sequencing determined that the patient is compound heterozygous for p.Arg341His and p.Arg370Trp CARS1. Yeast complementation assays showed that the p.Arg341His variant has a hypomorphic effect and that the p.Arg370Trp variant causes a complete loss-of-function effect. CONCLUSION: This study is the second report of pathogenic CARS1 variants and expands the allelic and phenotypic heterogeneity of CARS1-associated disease.

Humans↗
PubMed2026

CARS1 as a Prognostic Biomarker and Candidate Therapeutic Vulnerability in Hepatocellular Carcinoma: Insights Into Tumor Progression and the Immune Microenvironment.

BACKGROUND: Cysteinyl-tRNA synthetase 1 (CARS1) has been included in ferroptosis-related prognostic signatures, but its clinicopathological relevance, cellular functions, and relationship with the immune microenvironment in hepatocellular carcinoma (HCC) remain incompletely characterized. METHODS: Transcriptomic and clinical data from The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset were integrated with corresponding data from an institutional HCC tissue cohort of 60 patients. CARS1 expression was evaluated by immunohistochemistry, and immune infiltration was examined using single-sample gene-set enrichment analysis (ssGSEA) and multiplex immunofluorescence, as well as by analyzing public single-cell datasets. The effects of CARS1 depletion were evaluated in MHCC97H and Hep3B cells using Cell Counting Kit-8 (CCK-8) assays, cell-cycle profiling, wound-healing assays, Transwell migration assays, western blotting, and erlotinib-sensitivity assays. RESULTS: CARS1 expression was elevated in HCC and was associated with adverse clinicopathological features and poor overall survival. Quantitative immunohistochemistry confirmed elevated CARS1 protein expression in tumor tissues. CARS1 depletion inhibited cell proliferation, altered cell-cycle distribution, impaired migration, and enhanced in vitro sensitivity to erlotinib. High CARS1 expression was also associated with increased infiltration of Th2-like immune cells. CONCLUSIONS: Elevated CARS1 expression is associated with an adverse biological and immune phenotype in HCC. These clinical, histopathological, and loss-of-function findings support further investigation of CARS1 as a prognostic marker and candidate therapeutic target in HCC, although additional mechanistic and in vivo validation is required.

Humans↗
PubMed1976

Role of lysyl-tRNA in the regulation of lysine biosynthesis in Escherichia coli K12.

A mutant of lysyl-tRNA synthetase has been isolated in Escherichia coli K12. With this strain the Kmapp for lysine is 25 fold higher than with the parental strain. The percentage of charged tRNAlys in vivo is only 7 per cent (as against 65 per cent with HFR H). Under these conditions no derepression of synthesis is observed for three lysine biosynthetic enzymes (AK III, ASA-dehydrogenase, DAP-decarboxylase) ; a partial derepression is obtained in the case of the dhdp-reductase. Thus lysyl-tRNA does not act as the only corepressor molecule in the lysine regulon.

Amino Acyl-tRNA Synthetases↗
PubMed2025

Aminoacyl-tRNA Specificity of a Ligase Catalyzing Non-ribosomal Peptide Extension.

Peptide aminoacyl-transfer ribonucleic acid ligases (PEARLs) are amide-bond-forming enzymes that extend the main chain of peptides by using aminoacyl-tRNA (aa-tRNA) as a substrate. In this study, we investigated the substrate specificity of the PEARL BhaBCAla from Bacillus halodurans, which utilizes Ala-tRNAAla. By leveraging flexizyme, a ribozyme capable of charging diverse acids onto a desired tRNA, we generated an array of aa-tRNAs in which we varied both the amino acid and the tRNA to dissect the substrate scope of BhaBCAla. We demonstrate that BhaBCAla catalyzes peptide extension with noncognate proteinogenic and noncanonical amino acids, hydroxy acids, and mercaptocarboxylic acids when attached to tRNAAla. For most of these, the efficiency was considerably reduced compared to Ala, indicating that the enzyme recognizes the amino acid. By variation of the different parts of the tRNA, enzyme specificity was shown to also depend on the acceptor stem and the anticodon arm of the tRNA. These findings establish the molecular determinants of PEARL specificity and provide a foundation for engineering these enzymes for broader applications in peptide synthesis.

RNA, Transfer, Amino Acyl↗
PubMed1976

Affinity labelling of phenylalanyl-tRNA synthetase from E. coli MRE-600 by E. coli tRNAphe containing photoreactive group.

The photoinduced reaction of phenylalanyl-tRNA synthetase (E.C.6.1.1.20) from E.coli MRE-600 with tRNAphe containing photoreative p-N3-C6H4-NHCOCH2-group attached to 4-thiouridine sU8 (azido-tRNAphe) was investigated. The attachment of this group does not influence the dissociation constant of the complex of Phe-tRNAphe with the enzyme, however it results in sevenfold increase of Km in the enzymatic aminoacylation of tRNAphe. Under irradiation at 300 nm at pH 5.8 the covalent binding of [14C]-Phe-azido-tRNAphe to the enzyme takes place 0.3 moles of the reagent being attached per mole of the enzyme. tRNA prevents the reaction. Phenylalanine, ATP,ADP,AMP, adenosine and pyrophosphate (2.5 xx 10(-3) M) don't affect neither the stability of the tRNA-enzyme complex nor the rate of the affinity labelling. The presence of the mixture of either phenylalanine or phenylalaninol with ATP as well as phenylalaninol adenylate exhibits 50% inhibition of the photoinduced reaction. Therefore, the reaction of [14C]-Phe-azido-tRNA with the enzyme is significantly less sensitive to the presence of the ligands than the reaction of chlorambucilyl-tRNA with the reactive group attached to the acceptor end of the tRNA studied in 1. It has been concluded that the kinetics of the affinity labelling does permit to discriminate the influence of the low molecular weight ligands of the enzyme on the different sites of the tRNA enzyme interaction.

Adenine Nucleotides↗