Fig. 8: Proposed kinetic model for the binding process of the Mtb-ileS T-box to tRNA.
From: Translational T-box riboswitches bind tRNA by modulating conformational flexibility
In the absence of tRNA, the T-box samples both an open and a closed conformation (left, blue background). In the presence of tRNA, the T-box binds tRNA through a two-step process where interactions with the anticodon are established first, forming a partially bound state (middle, yellow background). The partially bound state exhibits conformational heterogeneity revealed by the WT T-box/ tRNAIle-ΔNCCA complex. In the One-state subpopulation (middle, top), tRNAIle-ΔNCCA transiently binds, leading to a 0.5 FRET state, with a 6.4 s lifetime (τ(0.5)partially_1) before dissociating. In the Two-state subpopulation (middle, bottom), tRNAIle-ΔNCCA remains stably bound, and rapidly fluctuates between a 0.5 FRET state (with a lifetime of τ(0.5)partially_2) and a 0.7 FRET state (with a lifetime of τ(0.7)partially). The 0.7 FRET state in the partially bound state resembles the 0.7 FRET state in the fully bound state (right, green background), but with a much shorter lifetime due to the absence of the NCCA-discriminator interactions. In the presence of an intact, uncharged NCCA end, the NCCA end of tRNA engages with the discriminator domain, leading to a long-lived fully bound state (right, green background, τ(0.7)fully). Rate constants (blue) are shown for the different binding states. Rate constants for the binding and dissociation during anticodon recognition step and for the conformational fluctuations within the Two-state subpopulation of the partially bound state are determined from the experiments using WT T-box riboswitch and tRNAIle-ΔNCCA at a tRNA concentration of 100 nM. The lifetimes of all FRET states are shown in pink and are calculated from dwell time analysis (Supplementary Table 6). The lifetimes of different FRET states in the partially bound state are determined from the experiments using WT T-box riboswitch and tRNAIle-ΔNCCA. The lifetime of the fully bound state is determined from the experiments using WT T-box riboswitch and tRNAIle. A detailed explanation of how the rates and lifetimes were assigned is given in Supplementary Fig. 8.
