The terminal adenosine of tRNA(Gln) mediates tRNA-dependent amino acid recognition by glutaminyl-tRNA synthetase.
Sequence-specific interactions between Escherichia coli glutaminyl-tRNA synthetase and tRNA(Gln) have been shown to determine the apparent affinity of the enzyme for its cognate amino acid glutamine during aminoacylation. Specifically, structural and biochemical studies suggested that residues Asp66, Tyr211, and Phe233 in glutaminyl-tRNA synthetase could potentially facilitate cognate amino recognition through their specific interactions with both A76 of tRNA(Gln)++ and glutamine. These residues were randomly mutated and the resulting glutaminyl-tRNA synthetase variants were screened in vivo for changes in their ability to recognize noncognate tRNAs and retention of tRNA-glutaminylation activity. When the variants selected in this way were characterized in vitro, they all showed dramatic decreases in apparent affinity (KM) for glutamine but little or no change in cognate tRNA affinity. Conservative replacements such as Y211F, F233L, and D66E resulted in 60-, 19-, and 18-fold increases compared to wild-type in the KM for glutamine, respectively, but had little effect on the turnover number (kcat). Nonconservative replacements affected both KM for glutamine and kcat; Y211S, F233D, and D66F displayed 1700, 3700, and 1200-fold decreases in kcat/KM for glutamine compared to wild-type. Double mutant cycle analysis indicated that Tyr211, and Phe233 interact strongly to enhance glutamine binding. These data now show that Asp66, Tyr211 and Phe233 mediate tRNA-dependent cognate amino acid recognition via the invariant 3'-terminal adenosine of tRNA(Gln).
Liu
J
J
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA.
Ibba
M
M
Hong
K W
KW
Söll
D
D
D000241:Adenosine / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D012351:RNA, Transfer, Gln / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D012351:RNA, Transfer, Gln; D014443:Tyrosine; D010649:Phenylalanine; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase; D000241:Adenosine