Fig. 4: Three-color smFRET spectroscopy of tRNA ASL binding to the T-box RNA.

a Representative binned (50 ms bin time) donor (AF488, blue), acceptor 1 (AF594, orange), and acceptor 2 (CF680R, red) fluorescence (upper) and FRET efficiency trajectories (lower). The FRET efficiency is defined as the fraction of total acceptor photons (acceptor 1 and acceptor 2), which are corrected for background, leaks, γ-factors, and direct excitation of acceptors (see Methods). Due to incomplete acceptor 2 labeling on the ASL, the bound state can appear as both 2- (binding to unlabeled ASL) and 3-color (binding to labeled ASL) segments as indicated by green and red asterisks above the FRET trajectories, respectively. After dissociation of ASL, in most cases, T-box molecules come back to the unbound state with the FRET efficiency values similar to those before binding (i.e., same unbound state) unless infrequent changes occur either in the unbound state (red arrows) or in the bound state (between the two blue arrows), indicating the presence of long-lasting memory. b Transition density map analysis. Correlation of the efficiency (i.e., fraction of total acceptor photons) values of the unbound T-box before ASL binding and after its dissociation (i.e., E values of two unbound states separated by a bound state). The distribution appears along the diagonal line for almost all the cases of the open (U1) state, and most cases of semi-open (U2) states, indicating the memory of the unbound conformation persists through the bound state. Conversely, the memory of the closed state (U3) is frequently lost in the bound state (transitions in the blue-shaded off-diagonal regions). c Kinetic model for tRNA ASL binding to the T-box RNA showing rates of interconversion (as lifetimes) between the different states (three bound states B1–B3 and three unbound states U1–U3) in the presence of 20 nM tRNA-ASL (see Supplementary Fig. 8 for the complete kinetic model accounting for incomplete labeling of acceptor 2). The area of the circle is proportional to the population of each state, and the thickness of the arrows is proportional to the rate. *binding rates with 20 nM ASL. Source data are provided as a Source Data file.