Specific Structural Elements of the T-Box Riboswitch Drive the Two-Step Binding of the tRNA Ligand

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 use small molecules as their ligands, as do most riboswitches, T-box riboswitches uniquely bind tRNAs and sense their aminoacylated states. The Stem I, located in the 5′ portion of the T-box, recognizes and interacts with the anticodon and elbow regions of the tRNA. The anti-terminator sequence, located in the 3′ portion of the T-box, senses the aminoacylation state by directly interaction with the NCCA sequence at the tRNA 3′ end. However, the kinetic trajectory that describes how these interactions are temporally established during tRNA binding remains unclear. Using single-molecule fluorescence resonance energy transfer (smFRET), we demonstrate that tRNA binds to the T-box in two steps, first anticodon recognition followed by sensing of the 3′ NCCA end. The second step is accompanied by an inward motion of the 3′ portion of the T-box riboswich relative to Stem I. By introducing point mutations to the T-box, 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