Solution NMR determination of hydrogen bonding and base pairing between the glyQS T box riboswitch Specifier domain and the anticodon loop of tRNAGly

•A T box riboswitch Specifier domain–tRNA anticodon arm complex was prepared.•The complex mirrors ribosomal codon–anticodon pairing with three base pairs.•Specifier bases rotate towards the minor groove to engage the tRNA anticodon.•The structure of the anticodon loop evolves to adopt a U-turn motif in the complex.•Loss of the U-turn reduces affinity but does not prevent complex formation. In Gram-positive bacteria the tRNA-dependent T box riboswitch regulates the expression of many amino acid biosynthetic and aminoacyl-tRNA synthetase genes through a transcription attenuation mechanism. The Specifier domain of the T box riboswitch contains the Specifier sequence that is complementary to the tRNA anticodon and is flanked by a highly conserved purine nucleotide that could result in a fourth base pair involving the invariant U33 of tRNA. We show that the interaction between the T box Specifier domain and tRNA consists of three Watson–Crick base pairs and that U33 confers stability to the complex through intramolecular hydrogen bonding. Enhanced packing within the Specifier domain loop E motif may stabilize the complex and contribute to cognate tRNA selection.