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Query: EC:6.1.1.10 (methionyl-tRNA synthetase)
 387 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Treatment of Escherichia coli formylmethionine tRNA with 2 M sodium bisulfite, pH 7.0, in 10 mM MgCl2 at 25 degrees results in formation of uridine/bisulfite adducts at U18 in the dihydrouridine loop, U37 in the anticodon, and U48 in the variable loop. Two products, corresponding to the two diastereoisomers of 5,6-dihydrouridine-6-sulfonate, are formed at each reactive site in the tRNA. Although none of the modifications cause complete loss of methionine acceptor activity, the modified tRNA is amino-acylated at a reduced rate and has a decreased affinity for E. coli methionyl-tRNA synthetase. Aminoacylation of [35S]bisulfite-labeled tRNAfMet with a limiting amount of purified enzyme followed by separation of the acylated and unacylated molecules and structural analysis has shown that the presence of a specific diastereoisomer of the uridine/bisulfite adduct in the anticodon base U37 alters the kinetic parameters for aminoacylation of tRNAfMet.
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PMID:Alteration of the kinetic parameters for aminoacylation of Escherichia coli formylmethionine transfer RNA by modification of an anticodon base. 1 33

Cysteinyl- and methionyl-tRNA synthetases (EC 6.11.-) were purified 1200- and 1000-fold, respectively, from sonic extracts of Paracoccus denitrificans strain 8944, and kinetics, substrate specificity and regulatory properties were determined using the ATP-PPi exchange reaction. Both enzymes had pH optima of approx. 8 and were inhibited by sulphydryl-group reagents. Cysteinyl-tRNA synthetase catalysed L-selenocysteine- and alpha-aminobutyric acid-dependent ATP-PPi exchange and methionyl-tRNA synthetase catalysed L-homocysteine-, L-selenomethionine- and norleucine-dependent ATP-PPi exchange. Both enzymes were inhibited by O-acetylserine. Cysteinyl-tRNA synthetase activity was stimulated by methionine and methionyl-tRNA synthetase activity was stimulated by sulphide, cysteine, and cysteic acid.
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PMID:Sulphur metabolism in Paracoccus denitrificans. Purification, properties and regulation of cysteinyl-and methionyl-tRNA synthetase. 1 93

Methionyl-tRNA synthetase from Escherichia coli can react with periodate-treated tRNA to form a Schiff's base through the epsilon-amino group of a lysine within the enzymic active center and the 2',3'-aldehyde groups created at the 3'-terminal ribose of tRNA. At alkaline pH, the Schiff's base equilibrium can be continuously and specifically displaced by reduction in situ with sodium cyanohydridoborate, which on the other hand leaves intact the reacting aldehyde groups of oxidized tRNA. The effects of temperature, pH and of reducing agent concentration on the rate and extent of reduction of the Schiff's base are analysed. Conditions are described (37 degrees C, pH 8.0, in the presence of 1 mM cyanohydridoborate) which allowed rapid and complete conversion of the monomeric trypsin-modified methionyl-tRNA synthetase into its 1:1 covalent complex with tRNAfMet.
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PMID:Complete inactivation and labeling of methionyl-tRNA synthetase by periodate-treated initiator tRNA in the presence of sodium cyanohydridoborate. 4 39

The 133,000 X g supernatant fraction prepared from ascites cells in 20 mM KCl (low CKl supernatant) contained the initiation factors EIF-1 and EIF-2 (and the elongation factore EF-1 and EF-2) but lacked EIF-3; thus, low KCl supernatant could be used to assay for EIF-3. EIF-3 was prepared from a crude initiation factor perparation (a 250 mM KCl extract of ascites cell ribosomes precipitated with 70% saturated ammonium sulfate) by chromatography on DEAE-Sephadex A-50 and hydroxylapatite. The EIF-O had no detectable EIF-1 and little or no EIF-2. Factor EIF-3 was required fro translation of encephalomyocarditis virus RNA. The molecular weight of EIF-3 was estimated by Sephadex G-200 filtration to be 139,000; the sedimentation coefficient was calculated to be about 5.8. EIF-3 formed a binary complex specifically with the initiator tRNA, Met-tRNAf, and if GTP was present the factor formed a ternary complex (EIF-3-Met-tRNAf-GTP). The EIF-3 preparation had no methionyl-tRNA synthetase activity to account for binding. Complex-formation was with eukaryotic Met-tRNAf and no other aminoacyl-tRNA. The binary and ternary complexes were retained quantitatively on Millipore filters (which was the most convenient assay), but they could also be demonstrated by filtration through Sephadex G-100 or by glycerol gradient centrifugation. GTP increased the rate, the amount, and the stability of complex formed; the ration of GTP to Met-tRNAf in the ternary complex appeared to be 1. The binary and the ternary complexes transferred Met-tRNAf to the 40 S ribosomal subunits, but not to 60 S subparticles. The factor-dependent binding of Met-tRNAf to the 40 S subunit did not require mRNA (or GTP). In the presence of 60 S subunits, the initiator tRNA bound to 40 S subunits was not transferred to 80 S ribosomes even if mRNA was added--that reaction may require another initiation factor. Treatment of EIF-3 with N-ethylmaleimide led to loss of its activity in complex formation and in support of the translation of encephalomyocarditis virus RNA. In addition to forming the binary and ternary complexes, and supporting the translation of encephalomyocarditis virus RNA, EIF-3 also increases the number of free ribosomal subunits by either preventing their association or causing dissociation of 80 S couples.
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PMID:Preparation and characterization of eukaryotic initiation factor EIF-3. Formation of binary (EIF-3-Met-tRNAf) and ternary (EIF-3-Met-tRNAf-GTP) complexes. 17 48

The charging of tRNA-Met-f and tRNA-Met-m in vivo and in vitro and initiation of polysomes during methionine limitation were studied in two strains of Escherichia coli K12. In the wild-type strain the distribution of polysomes as well as the kinetic parameters of methionyl-tRNA synthetase indicate preferential acylation of tRNA-Met-f. This preferential charging of tRNAM-et-f does not take place in a mutant strain which is also defective in initiation of polysomes during methionine limitation.
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PMID:Preferential charging of tRNA-Met-f in Escherichia coli K12. 21 45

Both the aminoacylation and isotopic ATP-PPi exchange activities of native and trypsin-modified methionyl-tRNA synthetases from Escherichia coli are specifically inactivated by incubation in the presence of periodate-treated initiator tRNA Met. The inactivation proceeds through the formation of a reversible Schiff's base between the epsilon-amino group of a lysine within the catalytic center of the enzyme and the 2',3'-aldehyde groups created at the 3'-terminal ribose of tRNA. The Schiff's base may be stabilized by reduction with sodium borohydride. Intact tRNA Met f competes with the inactivation by its dialdehyde. It has been verified in the case of the modified enzyme that the protection is afforded according to an equilibrium constant identical to that for tRNA Met f binding at the active site of the enzyme. Finally it is shown that the incorporation of one molecule of the dialdehyde of [14C]tRNA completely destroys the activity of the monomeric trypsin-modified methionyl-tRNA synthetase.
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PMID:Methionyl-tRNA synthetase from Escherichia coli. Inactivation and labeling by periodate-treated initiator tRNA. 22 89

The mesl- mutants of Saccharomyces cerevisiae cease division and accumulate in the G1 interval of the cell cycle when deprived of methionine or shifted from 23 to 36 degrees C in the presence of methionine. Synchronous cell cycle arrest results from a deficiency of charged methionyl-transfer ribonucleic acid (methionyl-tRNAMet) as shown by direct measurement of the in vivo pools of methionine, S-adenosylmethionine, and methionyl-tRNAMet. The deficiency of methionyl-tRNAMet in these cells is the consequence of a lesion in a single gene, mes1. mes1 appears to be the structural gene for the methionyl-tRNA synthetase because some revertants of this mutation exhibited a thermolabile methionyl-tRNA synthetase in vitro. A sufficient hypothesis to explain these and previous results is that the control of cell division by S. cerevisiae in response to nutrient limitation is mediated through aminoacyl-tRNA or subsequent steps in protein biosynthesis.
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PMID:Methionyl-transfer ribonucleic acid deficiency during G1 arrest of Saccharomyces cerevisiae. 32 18

The mechanism of the recognition of methionine by Escherichia coli methionyl-tRNA synthetase was examined by a kinetic study of the recognition of methionine analogues in the ATP-PPi exchange reaction and the tRNA-aminoacylation reaction. The results show that the recognition mechanism consists of three parts: (1) the recognition of the size, shape and chemical nature of the amino acid side chain at the methionine-binding stage of the reaction; (2) the recognition of the length of the side chain at the stage of aminoacyl-adenylate complex-formation; (3) the recognition of the sulphur atom in the side chain at the stage of methionyl-tRNA formation. It is proposed that the sulphur atom interacts with the enzyme to induce a conformational change. A model of the active site incorporating the mechanism of methionine recognition is presented.
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PMID:The aminoacylation of transfer ribonucleic acid. Recognition of methionine by Escherichia coli methionyl-transfer ribonucleic acid synthetase. 33 37

Binding of tRNA(Met/f) to the monomeric trypsin-modified methionyl-tRNA synthetase turns off the methionine-dependent isotopic ATP--PPi exchange. In the case of the dimeric native methionyltRNA synthetase, one anticooperatively bound tRNA(Met/f) inhibits the exchange by only 50%. These behaviours of tRNA do not require the integrity of the 3'-terminal adenosine. Esterification by methionine of the 3' end of tRNA reinforces the affinity of tRNA(Met/f)for the enzymes. In the case of the native enzyme, due to this effect, a second binding mode for methionyl-tRNA may be demonstrated through the isotopic exchange. This additional binding of tRNA corresponds to the expression of the anticooperatively blocked tRNA binding site. Methionine reverses competitively the reinforcing effect of the esterified methionyl moiety on tRNA binding. It is concluded that after esterification of tRNA, the aminoacyl residue still binds the enzyme, probably within the methionine activating site. The latter behaviour may account for the observation that excess methionine accelerates the aminoacylation turnover rate of tRNA(Met/f).
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PMID:Interrelation between transfer RNA and amino-acid-activating sites of methionyl transfer RNA synthetase from Escherichia coli. 33 59

Photoinduced covalent cross-linking has been used to identify a common surface of four methionine-accepting tRNAs which interact specifically with the Escherichia coli methionine:tRNA ligase (EC 6.1.1.10). tRNA--ligase mixtures were irradiated, and the covalently linked complexes were isolated and digested with T1 RNase (Schimmel & Budzik, 1977). The fragments lost from the elution profile of the T1 RNase digest were considered to have been cross-linked to the protein and therefore in intimate contact with the enzyme. Only specific cognate tRNA--ligase pairs produce covalently linked complexes. The four substrate tRNAs used in this study have substantially different sequences, but all showed a common cross-linking pattern, supporting the view that the sites cross-linked to the enzyme reflect the functionally common contact surface rather than particularly photoreactivity regions of tRNA. The cross-linked contact surface is comprised of three regions: (1) the narrow groove of the anticodon stem and its extension into the anticodon loop; (2) the 3' terminal residues; and (3) the 3' side of the "T arm". Unlike previous studies with other tRNAs, the D arm is not involved and significant radiation damage is suffered by the tRNA which must be taken into account in the analysis. The results are consistent with and complement chemical modification studies [Schulman, L. H., & Pelka, H. (1977) Biochemistry 16, 4256].
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PMID:Photocross-linking analysis of the contact surface of tRNA Met in complexes with Escherichia coli methionine:tRNA ligase. 36 5


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