Formation Of leucyl-tRNA Research Articles

The [formula omitted] subunit of Aquifex aeolicus leucyl-tRNA synthetase is responsible for cognate tRNA recognition

The active form of the leucyl-tRNA synthetase from an extreme thermophile Aquifex aeolicus has a heterodimeric (α/β type) quaternary structure that is unique among class I aminoacyl-tRNA synthetases. In an attempt to clarify the individual roles of each subunit in the function of leucyl-tRNA synthetase, several elementary activities were separately measured using each of the subunits alone or the reconstructed α/β complex. It was found that the β subunit alone is capable of recognizing its cognate tRNA, while the leucyl-adenylate formation and the overall leucyl-tRNA formation are detected only when both of the subunit proteins coexisted.

Characterization of versilin-sensitive sites in self-sensitive producer and sensitive non-producer or unrelated organism.

Studies on self-sensitivity of producer mutant vs. sensitivity of non-producer parent and unrelated organism showed that versilin inhibited spore germination and sporulation in the self-sensitive producer mutant, non-producer parent Aspergillus versicolor N5 and the unrelated sensitive Trichophyton rubrum. Sporulation appeared to be more sensitive than spore germination. The inhibition of in vivo synthesis of protein was very marked, but inhibition of RNA and DNA was slight and moderate, respectively. Thus versilin was not specific in its action, but the principal sensitive site was protein synthesis, as further suggested by inhibition of polyU-directed in vitro synthesis of polyphenylalanine. The activation of leucine was unaffected, but the formation of leucyl-tRNA was severely inhibited in all three strains. The differences in sensitivities between the strains were the same, whether as whole cells or as cell-free extracts. Thus the nature of the sensitive site appeared to be identical in the self-sensitive producer and sensitive non-producer or unrelated organism.

Mycoversilin, a new antifungal antibiotic. III. Mechanism of action on a filamentous fungus Trichophyton rubrum.

Mycoversilin is active against filamentous fungi, being specifically inhibitory to Trichophyton rubrum, minimum inhibitory concentration being 15 micrograms/ml. Mycoversilin inhibits sporulation to the extent of 28.5% even at the growth inhibitory concentration whereas inhibition of spore germination requires higher concentration. It has no effect on radial growth. Further it shows no action either on the release of UV absorbing materials or on the respiration of T. rubrum. However, the antibiotic inhibits in vivo synthesis of protein fairly strongly, DNA moderately and RNA slightly at the minimum inhibitory concentration. Cell-free protein synthesis is also strongly inhibited, the site of action being the inhibition of leucyl-tRNA formation by the antibiotic which has no action on leucine activation.

Carcinogenic azo dyes modify the acceptance of transfer ribonucleic acid for some amino acids

Postmitochondrial supernatant fractions of rat liver were incubated with 11 carcinogenic and non-carcinogenic derivatives of 4-dimethylaminoazobenzene and tRNA was isolated from this material. Only initiator tRNA from preparations pretreated with carcinogenic compounds showed a significantly enhanced acceptance for methionine. This effect was dependent from the dose and time of preincubation. Also the formation of lysyl- and alanyl-tRNA was stimulated after pretreatment of postmitochondrial supernatant fractions with carcinogenic derivatives whereas the synthesis of glycyl-tRNA was only slightly affected. No correlations were found between the carcinogenic activity of azo dyes and the charging of tRNA with phenylalanine, isoleucine, valine and arginine. All derivatives tested significantly inhibited the formation of leucyl-tRNA, carcinogenic compounds being more effective in this respect than non-carcinogenic ones. No changes in the charging of tRNA with amino acids were found if cytosols in the absence of microsomes were pretreated with the compounds tested. Only the presumed metabolite of 4-dimethylaminoazobenzene, 4'-hydroxy-4-dimethylaminoazobenzene, enhanced significantly the charging of initiator tRNA if preincubated with cytosol or with pure tRNA. Parent carcinogenic azo dyes are apparently converted into active intermediates by microsomal enzymes present in post-mitochondrial supernatant fractions and these ultimate carcinogens react with initiator tRNA and some other tRNA species and modify their acceptance.

Activation of leucyl-tRNA formation in preparations from rat liver

Activation of leucyl-tRNA formation in preparations from rat liver

Effet de l'ion Mg 2+ sur la croissance des têtards de Discoglossus pictus (OTTH) et sur la capacite de leurs microsomes a synthetiser les proteines in vitro

1. 1. Inhibition of growth of tadpoles was induced by an excess or lack of Mg 2+ ions in the breeding medium (mineral solutions). It was correlated with a decrease in the ability of the isolated microsomes to synthesize proteins. 2. 2. The action of Mg 2+ ions added in vitro to the incubation system suggested that the inhibition of protein synthesis was due to an excess or a lack of microsomal Mg 2+ content. 3. 3. The comparative action of Mg 2+ and Mn 2+ ions has been studied. Mn 2+ ions were comparable to Mg 2+ ions but at concentrations ten- or hundredfold less. 4. 4. Preliminary experiments dealing with the action of Mg 2+ ions on the formation of leucyl-tRNA were made.

Leucyl-tRNA synthetase. Mechanism of leucyl-tRNA formation.

The binding of ATP, leucine and tRNA to leucyl-tRNA synthestase and the properties of the complexes are examined. The results show that the binding of leucine to the enzyme is dependent on the presence of ATP and that the first step of leucine activation is the formation of an enzyme. ATP complex. The affinity constant of the enzyme for ATP is 4.5 × 106M−1. The binding of ATP to leucyl-tRNA synthetase requires the presence of divalent cations.The stability of the enzyme. ATP complex depends on the nature of these ions, showing that they are included in the complex. This complex, when incubated with leucine, leads to the enzyme. leucyladenylate complex capable of transfering its leucine to tRNA. The results indicate that leucyl-tRNA synthetase has one site for ATP, one for leucine and one tRNA receptor-site. The analysis of the binding of tRNA to the enzyme shows that leucyl-tRNA synthetase can bind specifically rRNAILeu or tRNAIILeu (acetylated or unacylated) with the same affinity constant: KA= 108 M−1. The rate constants of the binding reaction are increased in the presence of ATP plus leucine, suggesting existence of interactions between the site of the enzyme for leucyladenylate and its tRNA receptor site. A cyclic sequential mechanism is proposed for the preferential mode of action of leucyl-tRNA-synthetase. These results were obtained with the native leucyl-tRNA synthetase (the EII form) capable of catalyzing all reactions leading to the formation of leucyl-tRNA. They were also repeated with the enzyme form EI which does not catalyze the transfer of leucine from the enzyme. leucyladenylate complex to tRNA. In spite of its inability to transfer leucine, this enzyme form binds all the substrates of EII with affinity constants identical to those of EII. With the Ei form the rate constants of the tRNA binding reaction are no longer modified in the presence of ATP plus leucine indicating the loss of interactions between the enzyme sites for leucyladenylate and for tRNA.

Leucyl-tRNA synthetase. Two forms of the enzyme: role of sulfhydryl groups.

The role of sulhydryl groups in the enzymatic properties of leucyl-tRNA synthetase was examined and related to the differences between two forms of the enzyme. The titration of sulfhydryl groups shows that two groups are masked in EIi. e the transfer-inactive forms, as compared to EIIi. e the fully active enzyme. Results of the inactivation of leucyl-tRNA synthetase (EII) by N-methylmalemide and p-chloromercuribenzoate suggest that one sulfhydryl group is rapidly reacting and is essential to the transfer of leucine to tRNA. The binding of one mol mercuribenzoate per mol leucyl-tRNA synthetase leads to an enzyme which is unaffected in its ATP-pyrophosphate exchange activity, which binds all substrates (ATP, leucine, tRNA) with the same affinity as the ative enzyme, even though such an enzyme is completely inactivated for the formation of leucyl-tRNA. The rate constants of the native enzyme (EII) for the binding of tRNA are increased by the presence of ATP plus leucine, whereas in the case of enzyme (EII) treated with ethylmaleimide or wercuribenzoate, these rates remain unaffected by the presence of ATP plus leucine as in the case of EI. This shows that some interactions between the site of the enzyme for leucyladenylate and its tRNA receptor site are lost in EI and in the treated EII. These results indicate a strong similarity between the properties of EI and those of treated EII and suggest that one of the two sulfhydryl groups which are masked in EI is the sulfhydryl group essential for transfer. However, while EI partially lacks specificity for leucine and catalyzes the ATP-pyrophosphate exchange also with isoleucine, valine and methionine, the treated EII enzyme remains specific for leucine. This suggests, as does the fact that treatment with mercuribenzoate or ethylmaleimide does not modify the chromatograhic properties of EII, that the difference in the sulfhydryl reactivity is not the only one between EI and EII.

Replacement of Mg 2+ by monovalent cations in aminoacyl transfer RNA formation

1. The effect of monovalent cations on aminoacyl-tRNA formation in the absence of added Mg 2+ has been studied. 2. In valyl-tRNA formation, all monovalent cations studied (NH + 4 Li +, Na + and K +) are stimulatory, with NH + 4 and Li + being the most effective. An optimum concentration of monovalent cations is between 100 and 200 mM. 3. In leucyl-tRNA formation, NH + 4, Li + or K + stimulates the reaction significantly. Not only the yield of leucyl-tRNA, but also the rate of the reaction is almost the same as those observed in the presence of an optimal amount of Mg 2+. 4. In isoleucyl-tRNA formation, only NH + 4 is stimulatory. 5. A synergistic effect of Mg 2+ and some monovalent cations is observed in valyl-, isolecyul- and phenylalanyl-tRNA formation. 6. In the presence of NH + 4, K + or Na +, PP i-ATP exchange and amino acid hydroxamate formation are not observed, while they do occur in the presence of Li +. 7. It is assumed that in some aminoacyl-tRNA formations certain monovalent cations can replace Mg 2+ either completely or partially.

The stimulatory effect of ammonium or potassium ions on the activity of leucyl-tRNA synthetase from Escherichia coli

NH 4 + at an optimal concentration of 0.005 M stimulated the activity of leucyl-tRNA synthetase ( l-leucine: tRNA ligase (AMP), EC 6.1.1.4) in Escherichia coli 6-fold. The activities of isoleucyl- and phenylalanyl-tRNA synthetases were stimulated to lesser extents, by factors of 2.9 and 1.5, respectively, whereas those of valyl-, glycyl-, lysyl-, alanyl-, and seryl-tRNA synthetases were not affected. Stimulation of the enzymatic activity by NH 4 + was shown in the formation of leucyl-tRNA, but not in the formation of leucyl hydroxamate, suggesting that the NH 4 + is involved in the attachment of the activated leucine to the tRNA molecules. K + can partially substitute for NH 4 + in the stimulatory effect of the latter on leucyl-tRNA synthetase. At an optimal concentration of 0.08 M, K + stimulated the enzymatic activity 3-fold. Na + or Li + was not effective at the same concentration.

Multiple fractions of leucyl-transfer ribonucleic acid synthetase activity from Escherichia coli

Multiple fractions of leucyl-tRNA synthetase ( l-leucine: tRNA ligase (AMP), EC 6.1.1.4) activity were obtained from Escherichia coli K-12 and E. coli K-10 using continuous elution disc gel electrophoresis. Two types of enzymatic specificities could be distinguished by charging experiments using a given amount of tRNA. One type of enzymatic activity catalyzes the formation of leucyl-tRNA from about half of the tRNA molecules specific for leucine. The other type of enzymatic activity charges all the leucyl-tRNA.