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

The availability of various amino acids is essential for the synthesis of proteins during translation. In bacteria, T-box riboswitches play a critical role in regulating the concentrations of amino acids by sensing and decoding the aminoacylation status of tRNAs for precise control of amino acid biosynthesis and transport pathways. We used computer 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 show that the T-box aptamer populates three distinct thermodynamic states – undocked, predocked, and docked – depending on the relative orientation between its three stems (stem I, II, and IIA/B). The relative population of the three states is strongly dependent on the Mg ²⁺ concentration. In the undocked and predocked states, the freely moving stem I catches the tRNA from the solution using the ‘fly-casting’ mechanism. Subsequently, the tRNA-bound stem I docks to stem II in a specific orientation with the help of noncanonical intra- and inter-backbone hydrogen bonds, which facilitate tRNA interaction with the discriminator domain to detect the aminoacylation state of tRNA and regulate the gene. Since T-box riboswitches are prime targets in designing antibiotics, our proposed mechanism of tRNA recognition by T-box reveals multiple critical sites in the riboswitch that can be targeted for developing new antibiotics. Interestingly, molecules acting as antibiotics exist targeting one of the proposed sites. These results also have broad implications for understanding the role of specific RNA-RNA interactions in various cellular processes.