THE TERMINAL ADENOSINE OF TRNA(GLN) MEDIATES TRANSFER-RNA-DEPENDENT AMINO-ACID RECOGNITION BY GLUTAMINYL-TRANSFER-RNA SYNTHETASE

Sequence-specific interactions between Escherichia coli glutaminyl-tRN A synthetase and tRNA(Gln) have been shown to determine the apparent a ffinity of the enzyme for its cognate amino acid glutamine during amin oacylation. 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 synth etase variants were screened in vivo for changes in their ability to r ecognize noncognate tRNAs and retention of tRNA-glutaminylation activi ty. When the variants selected in this way were characterized in vitro , they all showed dramatic decreases in apparent affinity (K-M) for gl utamine 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 K-M for glutamine, res pectively, but had little effect on the turnover number (k(cat)). Nonc onservative replacements affected both K-M for glutamine and k(cat); Y 211S, F233D, and D66F displayed 1700, 3700, and 1200-fold decreases in k(cat)/K-M for glutamine compared to wild-type. Double mutant cycle a nalysis 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 invarian t 3'-terminal adenosine of tRNA(Gln).