The 2-thiouridine (s 2 U) at the wobble position of certain bacterial and eukaryotic tRNAs enhances aminoacylation kinetics, assists proper codon–anticodon base pairing at the ribosome A-site, and prevents frameshifting during translation. By mass spectrometry of affinity-purified native Escherichia coli tRNA 1 Gln UUG , we show that the complete modification at the wobble position 34 is 5-carboxyaminomethyl-2-thiouridine (cmnm 5 s 2 U). The crystal structure of E. coli glutaminyl-tRNA synthetase (GlnRS) bound to native tRNA 1 Gln and ATP demonstrates that cmnm 5 s 2 U34 improves the order of a previously unobserved 11-amino-acid surface loop in the distal β-barrel domain of the enzyme and imparts other local rearrangements of nearby amino acids that create a binding pocket for the 2-thio moiety. Together with previously solved structures, these observations explain the degenerate recognition of C34 and modified U34 by GlnRS. Comparative pre-steady-state aminoacylation kinetics of native tRNA 1 Gln , synthetic tRNA 1 Gln containing s 2 U34 as sole modification, and unmodified wild-type and mutant tRNA 1 Gln and tRNA 2 Gln transcripts demonstrates that the exocyclic sulfur moiety improves tRNA binding affinity to GlnRS 10-fold compared with the unmodified transcript and that an additional fourfold improvement arises from the presence of the cmnm 5 moiety. Measurements of Gln–tRNA Gln interactions at the ribosome A-site show that the s 2 U modification enhances binding affinity to the glutamine codons CAA and CAG and increases the rate of GTP hydrolysis by E. coli EF-Tu by fivefold.