Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

Complementary and antiparallel oligonucleotides bind to exposed regions of the tRNA molecule. Aminoacylation in the presence of triplets has been used to determine the role of the anticodon in the interaction between methionyl-tRNA synthetase and initiator tRNA. ApUpG has no effect on the charging even when 70% of the tRNA is bound to the triplet, whereas in the presence of GpGpU which binds to the A-C-C sequence adjacent to the 3' terminal adenosine that fraction of the tRNA which is bound to the triplet is completely unavailable for charging. Hence the anticodon is probably not involved in a primary interaction while the A-C-C-A-OH clearly is. This conclusion is supported by the failure of the isolated anticodon loop and stem oligonucleotides to inhibit the aminoacylation reaction.
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PMID:The role of the anticodon in the interaction between methionyl-tRNA synthetase and bacterial initiator tRNA. 460 44

Ribonuclease T2, nuclease S1, and snake venom phosphodiesterase were used as a structural probe for investigation of the interaction between Escherichia coli tRNAfMet and methionyl-tRNA synthetase, and the cleavage sites were analyzed by a rapid sequencing gel electrophoresis of 5'-32P-labeled tRNA. Both endonucleases cleaved the D-loop of synthetase-bound tRNA much more extensively than that of the free tRNA. Positions of A14, G15, A22, and G23 in the D-loop and C35 in the anticodon of the synthetase-bound tRNA were more susceptible to RNase T2. The synthetase-bound tRNA was predominantly cleaved by nuclease S1 at position of G15, G19, G20, and G23 in the D-loop and G2 in the acceptor stem. In contrast, the synthetase-bound tRNA was more resistant to the 3'-exonuclease, snake venom phosphodiesterase, than was the free tRNA molecule. These results suggest conformational change of the tRNA by the synthetase binding which weakened tertiary interaction between the D-loop and T psi C-loop/extra-loop. Production of acid-soluble radioactivity was also examined in the limited digestion of 5'-32P-labeled tRNA or 3'-14C-labeled methionyl-tRNA. The synthetase enhanced the release of acid-soluble oligonucleotides from the 5'-end of the tRNA but suppressed that from the 3'-end of the molecule. These results are consistent with that obtained by gel electrophoresis.
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PMID:Methionyl-tRNA synthetase-induced conformational change of Escherichia coli tRNAfMet. 626 70

Both the tRNA aminoacylation and amino-acid-dependent ATP-PPi exchange activities of monomeric trypsin-modified methionyl-tRNA synthetase from sheep liver are lost upon incubation with oxidized initiator tRNAMet. The inactivation, which reflects the formation of a Schiff's base between the 5'-terminal adenosine of tRNA and a lysine within the catalytic site of the enzyme, is accompanied by the covalent attachment of one tRNA molecule per enzyme molecule. The affinity labeling method is applied to the sheep liver complex of Mr 10(6) carrying seven aminoacyl-tRNA synthetase activities, from which the monomeric trypsin-modified methionyl-tRNA synthetase (Mr 68 000) was derived. Upon incubation with oxidized initiator tRNAMet, the methionyl-tRNA synthetase activity of the complex is lost. Of the eleven polypeptide chains composing the high-molecular-weight complex, only one polypeptide chain with Mr 103 000 reacts with the modified tRNAMet. The blocking by periodate-treated tRNA of the methionyl-tRNA synthetase activity in the complex has no effect on the other aminoacyl-tRNA synthetase activities. This strongly argues in favor of the independent parallel functioning of the seven aminoacyl-tRNA synthetases associated in a high-molecular-weight complex.
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PMID:Macromolecular complex of aminoacyl-tRNA synthetases from sheep liver. Identification of the methionyl-tRNA synthetase component by affinity labeling. 628 5

Derivatives of E. coli tRNAfMet containing single base substitutions at the wobble position of the anticodon have been enzymatically synthesized in vitro. The procedure involves excision of the normal anticodon, CAU, by limited digestion of intact tRNAfMet with RNase A. RNA ligase is then used to join each of four trinucleotides, NAU, to the 5' half molecule and to subsequently link the 3' and modified 5' fragments to regenerate the anticodon loop. Synthesis of intact tRNAfMet containing the anticodon CAU by this procedure yields a product which is indistinguishable from native tRNAfMet with respect to its ability to be aminoacylated by E. coli methionyl-tRNA synthetase. Substitution of any other nucleotide at the wobble position of tRNAfMet drastically impairs the ability of the synthetase to recognize the tRNA. Measurement of methionine acceptance in the presence of high concentrations of pure enzyme has established that the rate of aminoacylation of the AAU, GAU and UAU anticodon derivatives of tRNAfMet is four to five orders of magnitude slower than that of the native or synthesized tRNA containing C as the wobble base. In addition, the inactive tRNA derivatives fail to inhibit aminoacylation of normal tRNAfMet, indicating that they bind poorly to the enzyme. These results support a model involving direct interaction between Met-tRNA synthetase and the C in the wobble position during aminoacylation of tRNAfMet.
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PMID:Base substitutions in the wobble position of the anticodon inhibit aminoacylation of E. coli tRNAfMet by E. coli Met-tRNA synthetase. 633 82

Using fluorescent antibody staining, we have established the association of methionyl-tRNA synthetase with the endoplasmic reticulum in PtK2 cells. After Triton X-100 extraction, 70% of the recovered aminoacyl-tRNA synthetase activity was found in the detergent-insoluble fraction. This fraction of the enzyme remained localized with insoluble endoplasmic reticulum antigens and with ribosomes, which were stained with acridine orange. By both fluorescence microscopy and electron microscopy the organization of the detergent-insoluble residue was found to depend on the composition of the extracting solution. After extraction with a microtubule-stabilizing buffer containing EGTA, Triton X-100, and polyethylene glycol (Osburn, M., and K. Weber, 1977, Cell, 12:561-571) the ribosomes were aggregated in large clusters with remnants of membranes. After extraction with a buffer containing Triton X-100, sucrose, and CaCl2 (Fulton, A. B., K. M. Wang, and S. Penman, 1980, Cell, 20:849-857), the ribosomes were in small clusters and there were few morphologically recognizable membranes. In both cases the methionyl-tRNA synthetase and some endoplasmic reticulum antigens retained approximately their normal distribution in the cell. Double fluorochrome staining showed no morphological association of methionyl-tRNA synthetase with the microtubule, actin, or cytokeratin fiber systems of PtK2 cells. These observations demonstrate that detergent-insoluble cellular components, sometimes referred to as "cytoskeletal" preparations, contain significant amounts of nonfilamentous material including ribosomes, and membrane residue. Caution is required in speculating about intermolecular associations in such a complex cell fraction.
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PMID:Association of methionyl-tRNA synthetase with detergent-insoluble components of the rough endoplasmic reticulum. 633 26

E. coli tRNAMetf was hydrolyzed with RNase A using a limited amount of the enzyme to give two half molecules lacking the anticodon trimer and 3'-terminal dimer. Chemically synthesized trimers CUAp and UUAp were joined to the 5'-half molecules by phosphorylation with polynucleotide kinase plus ATP followed by treatment with RNA ligase. These modified tRNAMetf species had anticodons complementary to the termination codons UAG and UAA. Two half fragments were joined by a similar procedure to yield a molecule lacking the anticodon trimer and the 3'-dimer. Methionine acceptor activity of these tRNA was tested under conditions in which the CAU inserted control tRNAMetf accepted methionine. It was found that all three modified molecules were not recognized by the methionyl-tRNA synthetase from E.coli. The other sixteen amino acids were not incorporated with partially purified aminoacyl-tRNA synthetases.
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PMID:Modification of the anticodon triplet of E.coli tRNAMetf by replacement with trimers complementary to non-sense codons UAG and UAA. 634 66

Previous work from our laboratory identified several specific sites in Escherichia coli tRNAfMet that are essential for recognition of this tRNA by E. coli methionyl-tRNA synthetase (EC 6.1.1.10). Particularly strong evidence indicated a role for the nucleotide base at the wobble position of the anticodon in the discrimination process. To further investigate the structural requirements for recognition in this region, we have synthesized a series of tRNAfMet derivatives containing single base changes in each position of the anticodon. In addition, derivatives containing permuted sequences and larger and smaller anticodon loops have been prepared. The variant tRNAs have been enzymatically synthesized in vitro. The procedure involves excision of the normal anticodon, CAU, by limited digestion of intact tRNAfMet with pancreatic RNase. This step also removes two nucleotides from the 3' CpCpA end. T4 RNA ligase is used to join oligonucleotides of defined length and sequence to the 5' half-molecule and subsequently to link the 3' and modified 5' fragment to regenerate the anticodon loop. The final step of the synthesis involves repair of the 3' terminus with tRNA nucleotidyltransferase. The synthetic derivative containing the anticodon CAU is aminoacylated with the same kinetics as intact tRNAfMet. Base substitutions in the wobble position reduce aminoacylation rates by at least five orders of magnitude. The rates of aminoacylation of derivatives having base substitutions in the other two positions of the anticodon are 1/55 to 1/18,500 times normal. Nucleotides that have specific functional groups in common with the normal anticodon bases are better tolerated at each of these positions than those that do not. A tRNAfMet variant having a six-membered loop containing only the CA sequence of the anticodon is aminoacylated still more slowly, and a derivative containing a five-membered loop is not measurably active. The normal loop size can be increased by one nucleotide with a relatively small effect on the rate of aminoacylation, indicating that the spatial arrangement of the nucleotides is less critical than their chemical nature. We conclude from these data that recognition of tRNAfMet requires highly specific interactions of methionyl-tRNA synthetase with functional groups on the nucleotide bases of the anticodon sequence.
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PMID:Anticodon loop size and sequence requirements for recognition of formylmethionine tRNA by methionyl-tRNA synthetase. 635 55

The three-dimensional structures of two animoacyl-tRNA synthetases, the methionyl-tRNA synthetase from Escherichia coli (MetRS) and the tyrosyl-tRNA synthetase from Bacillus stearothermophilus (TyrRS), show a remarkable similarity over a span of about 140 amino acids. The region of homologous folding corresponds to a five-stranded parallel beta-sheet, including a mononucleotide-binding fold. One cysteine and two histidine residues that were found to be invariant in the amino acid sequences occupy similar places in the nucleotide-binding fold. In TyrRS, these residues are close to the adenylate binding site, and in MetRS to the Mg2+-ATP binding site.
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PMID:Structural homology in the amino-terminal domains of two aminoacyl-tRNA synthetases. 636 12

In previous work we identified several specific sites in Escherichia coli tRNAfMet that are essential for recognition of this tRNA by E. coli methionyl-tRNA synthetase (MetRS) (EC 6.1.1.10). Particularly strong evidence indicated a role for the nucleotide base at the wobble position of the anticodon in the discrimination process. We have now investigated the aminoacylation activity of a series of tRNAfMet derivatives containing single base changes in each position of the anticodon. In addition, derivatives containing permuted sequences and larger and smaller anticodon loops have been prepared. The variant tRNAs have been enzymatically synthesized in vitro by using T4 RNA ligase (EC 6.5.1.3). Base substitutions in the wobble position have been found to reduce aminoacylation rates by at least five orders of magnitude. Derivatives having base substitutions in the other two positions of the anticodon are aminoacylated 55-18,500 times slower than normal. Nucleotides that have specific functional groups in common with the normal anticodon bases are better tolerated at each of these positions than those that do not. A tRNAfMet variant having a six-membered loop containing only the CA sequence of the anticodon is aminoacylated still more slowly, and a derivative containing a five-membered loop is not measurably active. The normal loop size can be increased by one nucleotide with a relatively small effect on the rate of aminoacylation, which indicates that the spatial arrangement of the nucleotides is less critical than their chemical nature. We conclude from these data that recognition of tRNAfMet requires highly specific interactions of MetRS with functional groups on the nucleotide bases of the anticodon sequence. Several other aminoacyl-tRNA synthetases are known to require one or more anticodon bases for efficient aminoacylation of their tRNA substrates, and data from other laboratories suggest that anticodon sequences may be important for accurate discrimination between cognate and noncoagnate tRNAs by these enzymes.
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PMID:Recognition of tRNAs by aminoacyl-tRNA synthetases: Escherichia coli tRNAMet and E. coli methionyl-tRNA synthetase. 638 81

A protein affinity labeling derivative of E. coli tRNAfMet has been prepared which carries an average of one reactive side chain per molecule, distributed over four structural regions. Each side chain contains a disulfide bond capable of reaction with cysteine residues and an N-hydroxysuccinimide ester group capable of coupling to lysine epsilon-amino groups in proteins. Reaction of the modified tRNA with E. coli methionyl-tRNA synthetase leads to crosslinking only by reaction with lysine residues in the protein. Examination of the tRNA present in the crosslinked complex reveals that the enzyme is coupled to side chains attached to the 5' terminal nucleotide, the dihydrouridine loop, the anticodon and the CCA sequence. Digestion of the crosslinked enzyme with trypsin followed by peptide mapping reveals that the major crosslinking reactions occur at four specific lysine residues, with minor reaction at two additional sites. Native methionyl-tRNA synthetase contains 90 lysine residues, 45 in unique sequences of the dimeric alpha 2 enzyme. Crosslinking of the protein to different regions in tRNAfMet thus occurs with the high degree of selectivity necessary for use in determining the peptide sequences which are near specific nucleotide sequences of tRNA bound to the protein.
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PMID:Modification of specific lysine residues in E. coli methionyl-tRNA synthetase by crosslinking to E. coli formylmethionine tRNA. 642 68


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