Abstract
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.