FIGURE 5.
Inhibition of ribosome recycling by AGs. (A) An in vitro assay for ribosome recycling. Dissociation of deacyl 3′-[32P]-tRNAPhe from the P site of m291-programmed ribosomes was measured in the absence or presence of various factors (as indicated). Stimulation of tRNA release required RRF, EF-G (GTP), and IF3, as reactions with single omissions showed no apparent catalysis. (B,C) Examples of experiments measuring the effect of Neo (various concentrations, as indicated) on tRNA release from control (B) or A1408G/G1491U (C) ribosomes in the presence of RRF, EF-G (GTP), and IF3. Fraction of radiolabeled tRNA bound was plotted against time, and the data were fit to a single exponential equation to obtain koff. (D,E) koff values were plotted as a function of concentration of Neo (D) and Tob (E). Data were fit to a modified dose response equation to generate IC50 values for control, A1408G, and A1408G/G1491U cases, respectively, as follows: Neo IC50 = 1.9 ± 0.3, 1.5 ± 0.2, 1.3 ± 0.2 µM; Tob IC50 = 23 ± 3, 20 ± 2, 18 ± 2 µM.