Conformational Pathways of Translational T-box Riboswitch Governing tRNA Recognition for Gene Regulation

Amino acid availability is crucial for protein synthesis. T-box riboswitches regulate amino acid levels in bacteria by sensing tRNA aminoacylation, ensuring precise control of the biosynthesis and transport pathways. We used coarse-grained molecular dynamics simulations to probe the mechanism of tRNA recognition by a translational T-box riboswitch using the iles T-box riboswitch as a model system. We showed that the T-box aptamer transitions between undocked, predocked, and docked states with Mg²⁺ concentration governing their relative abundance. In the undocked and predocked states, the freely moving stem I catches tRNA from the solution using the "fly casting" mechanism. The tRNA-bound stem I then docks to stem II in a specific orientation stabilized by noncanonical hydrogen bonds. This docking enables interaction with the discriminator domain to sense tRNA aminoacylation and regulate gene expression. As T-box riboswitches are key antibiotic targets, our proposed mechanism reveals critical druggable sites, and the results have broader implications for understanding RNA-RNA interactions in cellular regulation.