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.