High-affinity recognition of specific tRNAs by an mRNA anticodon-binding groove

T-box riboswitches are modular bacterial noncoding RNAs that sense and regulate amino-acid availability through direct interactions with tRNAs. Between the 5' anticodon-binding Stem I and 3' amino acid-sensing domains of most T-boxes lies the Stem II domain of unknown structure and function. Here, we report a 2.8-Å cocrystal structure of the Nocardia farcinica ileS T-box in complex with its cognate tRNA(Ile). The structure reveals a perpendicularly arranged ultrashort Stem I containing a K-turn and an elongated Stem II bearing an S-turn. Both stems rest against a compact pseudoknot, dock via an extended ribose zipper, and jointly create a binding groove specific to the anticodon of its cognate tRNA. Contrary to proposed distal contacts to the tRNA elbow region, Stem II locally reinforces the codon-anticodon interactions between Stem I and tRNA, achieving low-nanomolar affinity. This study illustrates how mRNA junctions can create specific binding sites for interacting RNAs of prescribed sequence and structure.