Figure 5. Neomycin alters tRNA dynamics on the ribosome in a bimodal fashion.
(a) smFRET traces obtained in the presence of increasing neomycin concentrations were idealized to a four-state hidden Markov model (Methods). Time-averaged classical-state occupancy is shown versus neomycin concentration along with standard errors calculated from 1,000 bootstrap samples. (b) Puromycin was delivered to ribosomal complexes containing tRNAfMet in the P-site and Cy3-DSP-Met-Phe-tRNAPhe in the A-site in the presence of increasing concentrations of neomycin. Fluorescence intensity decays with time as fluorescently labeled peptide is released from the surface (Methods; Supplementary Fig. 7). Decreased rates of puromycin reactivity indicate decreased hybrid-state occupancy. Mean relative rates of puromycin reactivity (n = 2, s.d. = 0.05) calculated from the total normalized change in fluorescence over a 10-min period are shown versus neomycin concentration.