Two-step binding kinetics of tRNAGly by the glyQS T-box riboswitch and its regulation by T-box structural elements

T-box riboswitches are cis-regulatory RNA elements that regulate mRNAs encoding for aminoacyl tRNA synthetases or proteins involved in amino acid biosynthesis and transport. Rather than using small molecules as their ligands, as do most riboswitches, T-box riboswitches uniquely bind tRNA and sense their aminoacylated state. Whereas the anticodon and elbow regions of the tRNA interact with Stem I, located in the 59 portion of the T-box, sensing of the aminoacylation state involves direct binding of the NCCA sequence at the tRNA 39 end to the anti-terminator sequence located in the 39 portion of the T-box. However, the kinetic trajectory that describes how each of these interactions are established temporally during tRNA binding remains unclear. Using single-molecule fluorescence resonance energy transfer (smFRET), we demonstrate that tRNA binds to the riboswitch in a two-step process, first with anticodon recognition followed by NCCA binding, with the second step accompanied by an inward motion of the 39 portion of the T-box riboswitch relative to Stem I. By using site-specific mutants, we further show that the T-loop region of the T-box significantly contributes to the first binding step, and that the K-turn region of the T-box influences both binding steps, but with a more dramatic effect on the second binding step. Our results set up a kinetic framework describing tRNA binding by T-box riboswitches and highlight the important roles of several T-box structural elements in regulating each binding step.