Fig. 2. Frameshifting-prone translocation with EF-G(Q507) mutants.

a Schematic of an EF-G–ribosome complex in CHI state (adapted from PDB 4W29⁴⁵). Pept-tRNA (magenta) and deacylated tRNA (blue) are in CHI states. The zoom-in shows residue Q507 at the tip of domain 4 of EF-G (green) in contact with the anticodon loop of pept-tRNA. The ribosome is shown in gray and the mRNA in black. b–d Contour plots showing the distribution of FRET values during translocation on slippery mRNA mediated by EF-G(Q507A) (b), EF-G(Q507N) (c), and EF-G(Q507D) (d). Histograms at the right show distribution of FRET values after synchronization to the first transition below FRET 0.5. Pie charts indicate percentage of smFRET traces showing prolonged fluctuations between CHI and P/P states (dark gray) during translocation. Data are from at least four independent experiments (N = 4 for Q507A, N = 5 for Q507N, and N = 9 for Q507D). e Transition frequency between FRET states during translocation on slippery mRNA with Q507 mutants and EF-G(wt) (Supplementary Fig. 2 and Table 1). After addition of EF-G, transitions between FRET 0.4 (CHI) and 0.2 (P/P) states are predominant. Shown are mean values with error bars representing the standard deviations. Data are from at least three independent experiments (N = 3 for PRE, N = 12 for wt, N = 4 for Q507A, N = 5 for Q507N, and N = 9 for Q507D). f Correlation between frameshifting efficiency and the fraction of ribosomes sampling CHI states during translocation on slippery mRNA (Supplementary Fig. 3). Frameshifting efficiencies were measured at 22 °C to match the conditions of the smFRET experiments. Shown are mean values with error bars representing the standard deviation. Black line indicates a linear fit with the slope of 1.3 ± 0.1, R² = 0.9982. Frameshifting efficiencies are from three independent experiments (N = 3). The percentage of traces with CHI states is derived from at least three independent experiments (N = 12 for wt, N = 4 for Q507A, N = 5 for Q507N, and N = 9 for Q507D).