Figure 3.

Effects of RRF and EF-G concentrations on the rate of RRF/EF-G-dependent splitting of vacant ribosomes and 70S·mXR7·tRNA^Phe complexes. (A, D) Vacant 70S ribosomes (0.2 μM) or (B, E) 0.2 μM 70S ribosomes pre-incubated with 0.4 μM mXR7 mRNA and 0.4 μM tRNA^Phe were rapidly mixed with the mixture containing different concentrations of RRF, 3 μM EF-G, 1 μM IF3 (A) or 2 μM IF3 (B) or with the mixture containing different concentrations of EF-G, 5 μM RRF, 1 μM IF3 (D) or 2 μM IF3 (E). All mixtures were prepared in LS4 buffer. (C, F) The splitting rate q plotted versus the concentration, [S], of RRF (C) or EF-G (F) was fitted to the Michaelis–Menten equation q=q_cat[S]/([S]+K_M(S))