Connecting anticodon recognition with the active site of Escherichia coli glutaminyl-tRNA synthetase.
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.
Weygand-Durasević
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I
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114.
Rogers
M J
MJ
Söll
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D
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D000926:Anticodon / Q000378:metabolism*; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D012351:RNA, Transfer, Gln / Q000378:metabolism*; D012641:Selection, Genetic; D013379:Substrate Specificity; D013489:Suppression, Genetic
D000926:Anticodon; D012351:RNA, Transfer, Gln; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase