Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.1.1.4 (leucyl-tRNA synthetase)
 297 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Alanyl- and leucyl-tRNA synthetases from baker's yeast were purified to homogeneity in the presence of the protease inhibitor phenylmethylsulfonyl fluoride. Both consist of single polypeptide chains of 118 000 and 125 000 daltons, respectively, as determined by polyacrylamide gel electrophoresis under denaturing conditions. The monomeric structure of leucyl-tRNA synthetase differs from the dimeric one obtained previously in the absence of protease inhibitors. This illustrates the sensitivity of the synthetases to proteolytic actions and indicates that native structures can only be obtained under optimal protecting conditions. Alanyl- and leucyl-tRNA synthetases differ with respect to pH optimum (6.5 and 8.5, respectively), Michaelis constant for amino acid (1 mM and 0.03, respectively) and in the rate-limiting step for the tRNA aminoacylation reaction. Whereas the catalytic step itself was rate-limiting for alanyl-tRNA synthetase, a step occurring after this was rate-limiting for leucyl-tRNA synthetase.
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PMID:Purification and some properties of alanyl- and leucyl-tRNA synthetases from baker's yeast. 701 9

The chloroplast leucyl-tRNA synthetase from Phaseolus vulgaris was purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose, hydroxylapatite, and phosphocellulose. Finally, the pure enzyme was obtained after affinity chromatography on Blue Sepharose CL-6B using specific elution with a pure Escherichia coli tRNALeu isoacceptor. The specific activity of chloroplast leucyl-tRNA synthetase (1550 units/mg) is the highest ever obtained for a higher plant aminoacyl-tRNA synthetase. The purified enzyme has an optimal pH of 9.0 and Km values are, respectively, 2.6 X 10(-6) M for unfractionated E. coli tRNA, 0.85 X 10(-6) M for E. coli tRNA5Leu, 1.8 X 10(-4) M for ATP, and 1.4 X 10(-5) M for L-leucine. Chloroplast leucyl-tRNA synthetase is a large monomer which has a Mr of 122,000 as determined by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate and urea and by gel filtration. Determination of Stokes radius, diffusion coefficient, and frictional ratio suggests that the enzyme structure is rather compact. The amino acid composition shows a relatively large proportion of apolar residues. Specific antibodies were raised in rabbits against the pure chloroplast leucyl-tRNA synthetase.
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PMID:Purification and properties of chloroplast leucyl-tRNA synthetase from a higher plant: Phaseolus vulgaris. 705 99

The regulation of the activity of transport System L for neutral amino acids has been investigated in Chinese hamster ovary cells. Incubation of the temperature-sensitive leucyl-tRNA synthetase mutant CHO-tsH1 at marginally permissive temperatures results in leucine-limited growth and increased transport of branched chain and aromatic amino acids. This temperature-dependent transport enhancement is restricted to transport System L and results in increased Vmax values of uptake of System L substrates with unchanged Km values of uptake. Several lines of evidence suggest that trans-stimulation by intracellular amino acids cannot account for the increased System L activity in CHO-tsH1. Other temperature-sensitive aminoacyl-tRNA synthetase mutants of the Chinese hamster ovary line show increased transport of the amino acid corresponding to the synthetase defect after incubation at elevated temperatures. Increased transport by System L occurs in CHO-S, the parental line of CHO-tsH1, after growth in limiting levels of leucine. The System L enhancement can be prevented by cycloheximide but not, at early times, by actinomycin D. We conclude that the activity of transport System L in Chinese hamster ovary cells is regulated by a mechanism which appears to act at the level of translation.
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PMID:Regulation of amino acid transport system L in Chinese hamster ovary cells. 706 59

A temperature-sensitive mutant of Chinese hamster ovary cells with an altered leucyl-tRNA synthetase fails to grow and to incorporate amino acids into protein properly at or near the non-permissive temperature. This mutant was used to determine whether cessation of growth at the elevated temperature affected elongation factor EF-1, since the activity of EF-1 is markedly lower in non-growing cells in stationary phase than in rapidly-growing cells in exponential phase. Cell-free extracts prepared from cells maintained at 39 degrees C for 24 h showed a marked decrease in the ability to translate natural mRNAs, compared to cells incubated at 34 degrees C. However, the ability to translate poly(U), which requires elongation factor EF-1 (and EF-2), was not affected. Analyses of activities involved in the initiation of protein synthesis and in the activation of amino acids revealed that, with the exception of leucyl-tRNA synthetase, the rest of the components required for translation also appeared to be relatively stable even after 24 h at the elevated temperature. The effects of elevated temperature on cell-free extracts were also investigated. The results were similar to those obtained with intact cells; that is, except for leucyl-tRNA synthetase which was rapidly inactivated in vitro at 39 degrees C, other aminoacyl-tRNA synthetases and translational components involved in chain initiation and elongation were relatively stable. Thus, no change in EF-1 activity was detected as a result of arrested cell growth, an inherent lability of the elongation factor, or metabolic degradation as a consequence of a rapid turnover rate in the absence of protein synthesis.
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PMID:The effect of cessation of growth on protein synthesis in a mutant of Chinese hamster ovary cells with a temperature-sensitive leucyl-tRNA synthetase. 708 71

Four Escherichia coli operons, the leuV operon which encodes tRNA(1Leu), the leuX operon which encodes tRNA(6Leu), the metT operon which encodes tRNA(3Leu), and the argT operon which encodes tRNA(1Leu), were examined for the stringent response induced by serine hydroxamate and for growth rate-dependent regulation. In nuclease protection assays, the leuV operon displayed the stringent response in response to leucine starvation, analog inhibition, and growth of a temperature-sensitive leucyl-tRNA synthetase mutant at nonpermissive temperatures. The leuV operon also exhibited the stringent response in multicopy plasmids. The promoters of all four leucyl operons were fused to the gene for beta-galactosidase and inserted into the chromosome by using bacteriophage lambda. All except the leuX promoter displayed growth rate-dependent regulation, consistent with the recent report that the concentration of tRNA(6Leu) actually decreases as growth rate increases. The leuV promoter fused to the beta-galactosidase gene showed a decrease in efficiency in the presence of extrachromosomal copies of rRNA genes. All chromosomal tRNA genes examined showed decreased transcriptional activity following a stringent response, but the leuX gene responded to a lesser extent (3-fold versus 10-fold or more) than the others. Primer extension analysis of this promoter showed little if any response to serine hydroxamate treatment, suggesting that multiple levels of control may exist or that promoter context effects are important in regulation.
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PMID:In vivo regulatory responses of four Escherichia coli operons which encode leucyl-tRNAs. 768 Mar 41

We have used the technique of somatic cell hybridization to study the regulation of the neutral amino acid transport system L in Chinese hamster ovary (CHO) cells. The cell line CHO-ts025C1 has a temperature-sensitive mutation in leucyl-tRNA synthetase. At the nonpermissive temperature of 39 degrees C, CHO-ts025C1 cells are unable to charge leucyl-tRNA and behave as though starved for leucine by increasing their system L transport activity two- to fourfold. From the temperature-sensitive cell line, we have isolated a regulatory mutant cell line, CHO-C11B6, that has constitutively elevated system L transport activity. The CHO-C11B6 cell line retains the temperature-sensitive leucyl-tRNA synthetase mutation, but growth of this cell line is temperature resistant because its increased system L transport activity leads to increased intracellular leucine levels, which compensate for the defective synthetase. Hybrid cells formed by fusion of the temperature-sensitive CHO-ts025C1 cells and the temperature-resistant CHO-C11B6 cells show temperature-sensitive growth and temperature-dependent regulation of leucine transport activity. These data suggest that the system L activity of CHO cells is regulated by a dominant-acting element that is defective or absent in the regulatory mutant CHO-C11B6 cell line.
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PMID:Evidence for a regulatory element controlling amino acid transport system L in Chinese hamster ovary cells. 789 Aug 12

Aminoacyl-tRNA synthetase mutants of Escherichia coli are resistant to amdinocillin (mecillinam), a beta-lactam antibiotic which specifically binds penicillin-binding protein 2 (PBP2) and prevents cell wall elongation with concomitant cell death. The leuS(Ts) strain, in which leucyl-tRNA synthetase is temperature sensitive, was resistant to amdinocillin at 37 degrees C because of an increased guanosine 5'-diphosphate 3'-diphosphate (ppGpp) pool resulting from partial induction of the stringent response, but it was sensitive to amdinocillin at 25 degrees C. We constructed a leuS(Ts) delta (rodA-pbpA)::Kmr strain, in which the PBP2 structural gene is deleted. This strain grew as spherical cells at 37 degrees C but was not viable at 25 degrees C. After a shift from 37 to 25 degrees C, the ppGpp pool decreased and cell division was inhibited; the cells slowly carried out a single division, increased considerably in volume, and gradually lost viability. The cell division inhibition was reversible when the ppGpp pool increased at high temperature, but reversion required de novo protein synthesis, possibly of septation proteins. The multicopy plasmid pZAQ, overproducing the septation proteins FtsZ, FtsA, and FtsQ, conferred amdinocillin resistance on a wild-type strain and suppressed the cell division inhibition in the leuS(Ts) delta (rodA-pbpA)::Kmr strain at 25 degrees C. The plasmid pAQ, in which the ftsZ gene is inactivated, did not confer amdinocillin resistance. These results lead us to hypothesize that the nucleotide ppGpp activates ftsZ expression and thus couples cell division to protein synthesis.
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PMID:Penicillin-binding protein 2 inactivation in Escherichia coli results in cell division inhibition, which is relieved by FtsZ overexpression. 840 46

In order to study how Escherichia coli leucyl-tRNA synthetase recognizes tRNA(Leu) and discriminates it from the other two class II tRNAs, tRNA(Ser) and tRNA(Tyr), various mutations were introduced into class II tRNA transcripts. The discriminator base A73, but not the anticodon sequence, was found to serve as a critical recognition element of tRNA(Leu). A base substitution at the invariant nucleotide A14, but not at any of the other nucleotides characteristic of the E. coli tRNA(Leu) isoacceptors among the three class II tRNAs, caused significantly damaged aminoacylation with leucine. A two base-pair deletion in the long variable arm also resulted in no significant decrease of activity. Transplanting the three tertiary elements characteristic of E. coli tRNA(Leu) (i.e. the location of the G18G19 sequence in the D-loop, the A15 U48 base-pair and the stem pairing pattern of the long variable arm) besides the discriminator base change introduced the leucine charging activity in terms of Vmax/Km, up to 0.1 of that for the normal sequence of tRNA(Leu) into both tRNA(Ser) and tRNA(Tyr). These results indicate that A73 and A14 (or its vicinity) are involved in recognition by leucyl-tRNA synthetase, and that several tertiary elements play a significant role in the discrimination of tRNA(Leu) from the other two class II tRNAs.
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PMID:Recognition nucleotides of Escherichia coli tRNA(Leu) and its elements facilitating discrimination from tRNASer and tRNA(Tyr). 851 Jan 45

Autoantibodies to five aminoacyl-tRNA synthetases have been reported, and all have been associated with a syndrome of myositis and interstitial lung disease. Four of these synthetases exist free in the cytoplasm, but the fifth, isoleucyl-tRNA synthetase (recognized by anti-OJ autoantibodies), is a component of the multi-enzyme complex containing at least seven synthetases. In an effort to better understand the origins of these antibodies, we examined sera from 11 patients with anti-OJ autoantibodies for evidence of reaction with other components of the complex. All sera showed a characteristic pattern of 10 proteins bands by immunoprecipitation from HeLa cell extract. 10 of 11 sera significantly inhibited isoleucyl-tRNA synthetase enzyme activity. Serum and IgG from four patients also inhibited leucyl-tRNA synthetase activity, and serum and IgG from two inhibited lysyl-tRNA synthetase. Immunoblotting experiments supported reaction of the two sera with lysyl-tRNA synthetase, and revealed additional reactivity of three sera with a 160-kD component believed to be glutaminyl-tRNA synthetase. Despite reaction of some sera with additional synthetases, the immunoprecipitated tRNA appeared the same with all sera, and functioned as tRNA(ile). While reaction with more than one synthetase was seen with some anti-OJ sera, all synthetases targeted by anti-OJ sera were components of the complex, rather than unassociated synthetases. These findings suggest that an initial autoantibody response against isoleucyl-tRNA synthetase was followed by extension to involve other components of the synthetase complex. These observations may have implications for understanding the generation of antisynthetase autoantibodies.
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PMID:Reaction of anti-OJ autoantibodies with components of the multi-enzyme complex of aminoacyl-tRNA synthetases in addition to isoleucyl-tRNA synthetase. 851 67

A number of yeasts of the genus Candida translate the standard leucine-CUG codon as serine. This unique genetic code change is the only known alteration to the universal genetic code in cytoplasmic mRNAs, of either eukaryotes or prokaryotes, which involves reassignment of a sense codon. Translation of CUG as serine in these species is mediated by a novel serine-tRNA (ser-tRNACAG), which uniquely has a guanosine at position 33, 5' to the anticodon, a position that is almost invariably occupied by a pyrimidine (uridine in general) in all other tRNAs. We propose that G-33 has two important functions: lowering the decoding efficiency of the ser-tRNACAG and preventing binding of the leucyl-tRNA synthetase. This implicates this nucleotide as a key player in the evolutionary reassignment of the CUG codon. In addition, the novel ser-tRNACAG has 1-methylguanosine (m1G-37) at position 37, 3' to the anticodon, which is characteristic of leucine, but not serine tRNAs. Remarkably, m1G-37 causes leucylation of the ser-tRNACAG both in vitro and in vivo, making the CUG codon an ambiguous codon: the polysemous codon. This indicates that some Candida species tolerate ambiguous decoding and suggests either that (i) the genetic code change has not yet been fully established and is evolving at different rates in different Candida species; or (ii) CUG ambiguity is advantageous and represents the final stage of the reassignment. We propose that such dual specificity indicates that reassignment of the CUG codon evolved through a mechanism that required codon ambiguity and that ambiguous decoding evolved to generate genetic diversity and allow for rapid adaptation to environmental challenges.
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PMID:The non-standard genetic code of Candida spp.: an evolving genetic code or a novel mechanism for adaptation? 940 14


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