FIGURE 3.

Pre-steady state measurements of ternary complex formation and dissociation: dependence on factor concentration. A, the time-dependent response in fluorescence intensity observed upon addition of saturating amounts (400 nm) of either EF-Tu (blue) or EF-Tu·EF-Ts (black) to Cy3-labeled Phe-tRNA^Phe (5 nm) and GTP (10 μm) or EF-Tu·EF-Ts in the absence of GTP (gray). Fitting the data (see “Experimental Procedures”) provided a quantitative measure of the apparent rate of ternary complex formation, k_app,1. A focused plot of the formation process is also shown (inset). B, measurements of k_app,1 as a function of either EF-Tu (open diamonds) or EF-Tu·EF-Ts (closed squares). The inset shows the linear fits of early factor titration data points. C, the time-dependent response in fluorescence intensity observed upon addition of saturating amounts of GDP (100 μm) to ternary complex preformed as described in A with either EF-Tu (blue) or EF-Tu·EF-Ts (black). Identical experiments were performed using ADP (100 μm) (gray). D, fitting to a single exponential function provided a quantitative measure of the off-rate of ternary complex formation, k_app,2, as a function of either EF-Tu (open diamonds) or EF-Tu·EF-Ts (closed squares) as described in C. E, similar disassociation experiments were performed at varying GTP concentrations. Addition of saturating GDP to ternary complex preformed with an excess of EF-Tu (open diamonds) or EF-Tu·EF-Ts (closed squares) and 5 nm Phe-tRNA^Phe in the presence of GTP (50 nm–500 μm). Apparent decay rates, k_app,2, were estimated by fitting to a single exponential function. F, GDP (100 μm) was delivered to ternary complex preformed with EF-Tu·EF-Ts in the absence (black) or presence (tan) of kirromycin. The rate of GDP mediated dissociation in the absence of kirromycin (k_app,2 = 0.28 s⁻¹) was found to be 14 times faster than in the presence of kirromycin (k_app,2 = 0.02 s⁻¹). Error bars represent the S.E. from three independent experiments.