Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.1.1.18 (glutaminyl-tRNA synthetase)
 231 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Wild-type Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) poorly aminoacylates opal suppressors (GLN) derived from tRNA(Gln). Mutations in glnS (the gene encoding GlnRS) that compensate for impaired aminoacylation were isolated by genetic selection. Two glnS mutants were obtained by using opal suppressors differing in the nucleotides composing the base pair at 3.70: glnS113 with an Asp-235-->Asn change selected with GLNA3U70 (GLN carrying G3-->A and C70-->U changes), and glnS114 with a Gln-318-->Arg change selected with GLNU70 (GLN carrying a C70-->U change). The Asp-235-->Asn change was identified previously by genetic selection. Additional mutants were isolated by site-directed mutagenesis followed by genetic selection; the mutant enzymes have single amino acid changes (Lys-317-->Arg and Gln-318-->Lys). A number of mutants with no phenotype also were obtained randomly. In vitro aminoacylation of a tRNA(Gln) transcript by GlnRS enzymes with Lys-317-->Arg, Gln-318-->Lys, or Gln-318-->Arg changes shows that the enzyme's kinetic parameters are not greatly affected by the mutations. However, aminoacylation of a tRNA(Gln) transcript with an opal (UCA) anticodon shows that the specificity constants (kcat/Km) for the mutant enzymes were 5-10 times above that of the wild-type GlnRS. Interactions between Lys-317 and Gln-318 with the inside of the L-shaped tRNA and with the side chain of Gln-234 provide a connection between the acceptor end-binding and anticodon-binding domains of GlnRS. The GlnRS mutants isolated suggest that perturbation of the interactions with the inside of the tRNA L shape results in relaxed anticodon recognition.
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PMID:Functional communication in the recognition of tRNA by Escherichia coli glutaminyl-tRNA synthetase. 750 18

Escherichia coli glutaminyl-tRNA synthetase (GlnRS) specifically recognizes nucleotides in the anticodon and acceptor stem of tRNA(Gln). Extensive conformational changes in the tRNA(Gln):GlnRS complex and requirement for tRNA in glutaminyl-adenylate formation suggests that accurate anticodon recognition is required for aminoacylation. A 17 amino acid loop in GlnRS (residues 476 to 492) that connects two beta-ribbon motifs was targeted for saturation mutagenesis as the motifs span the anticodon binding domain and extend to the active site. Opal suppressor tRNAs (GLN) derived from tRNA(Gln) are poor substrates for GlnRS, and compensating mutations in glnS (the structural gene for GlnRS) were selected by the ability of the mutant gene product to aminoacylate such a suppressor (GLNA3U70). A number of mutations in loop 476 to 492 were identified by genetic selection, and two of the GlnRS purified mutant enzymes showed elevated specificity constants (kcat/Km) for aminoacylation of a tRNA(Gln)-derived transcript with the opal (UCA) anticodon when compared with the wild-type enzyme. The specificity constants for the mutant enzymes with the cognate tRNA(Gln) transcript (anticodon CUG) were unchanged. Therefore, region 476 to 492 has been identified in communicating anticodon recognition with the active site at a distance of more than 30 A away, supporting a proposed model from the structure of the complex between tRNA(Gln):GlnRS. A previous study has identified residues that interact with the inside of the L-shaped tRNA as communicating accurate anticodon recognition. Therefore, at least two pathways of communication have been identified in the accurate recognition of tRNA by GlnRS.
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PMID:Connecting anticodon recognition with the active site of Escherichia coli glutaminyl-tRNA synthetase. 802 95