Figure 2. Kanamycin binding to the decoding site increases time-averaged occupancy of specific ribosome conformations.

The time-averaged occupancy (Supplementary Fig. 1) in all nonzero FRET states was measured for the surface immobilized population of intact 70S E. coli ribosomes at increasing concentrations of kanamycin. The FRET values between P-site tRNA^fMet(Cy3-s⁴U8) and A-site fMet-Phe-tRNA^Phe(Cy5-acp³U47) report directly on three specific conformational intermediates in the translocation pathway: the classical state (~0.55 FRET), the hybrid 1 (~0.39 FRET) state and the hybrid 2 state (~0.24 FRET). (a–d) Histograms are shown for wild-type (a), G2553C 23S (b), A1408G 16S (c) and G2553C 23S + A1408G 16S (d) ribosomal complexes in the presence of increasing kanamycin concentrations (black to red lines). Histograms were constructed by summing FRET values over the first 50 frames of each acquired movie over all nonzero FRET states and plotting the spline-interpolated data points.