FIGURE 2.

Detecting the interaction of tRNA^Phe with TruB by nitrocellulose filtration and absorbance measurements. (A) Nitrocellulose filtration to determine the dissociation constant of [³H]-labeled tRNA^Phe to catalytically inactive TruB D48N (K_D = 1.4 ± 0.3 μM). (B) Determination of the dissociation constant for the tRNA^Phe-TruB D48N interaction by measuring the change in absorbance at 260 nm (the increase in absorbance due to the increasing TruB D48N concentration was subtracted). Hyperbolic fitting yielded a K_D of 0.34 ± 0.06 μM. (C) Stopped-flow experiments detecting the change in absorbance at 260 nm upon rapidly mixing tRNA^Phe (0.75 μM final concentration) with TruB D48N (10 μM), TruB WT (10 μM), or no TruB (labeled tRNA). The time courses shown are averages of 8–12 individual time courses and were fitted to a one-exponential equation (smooth lines) to determine the apparent rate constants: k_app(WT) = 5.2 sec⁻¹; k_app(D48N) = 12.7 sec⁻¹. Note the logarithmic time scale of the time courses. (D) TruB WT concentration dependence of the apparent rates of individual time courses recorded by stopped-flow measurements as in C. The average apparent rate over all measured concentrations is 6.0 ± 1.8 sec⁻¹, as indicated by the straight line.