Fig. 4. A dynamic model of ArfB recruitment.

a Experimental assay to monitor binding of ArfB to the ribosome in real time. P + n ribosome complexes containing fluorescein-labeled fMetPhe-tRNA^Phe (P + n(Flu)) are mixed with quencher-labeled ArfB (ArfB(540Q)) and fluorescein quenching is monitored in real time in a stopped-flow apparatus. b Time courses of ArfB binding with fixed concentration of P + 9(Flu) (0.015 µM) and increasing concentrations of ArfB(540Q) (0.05-0.5 µM) (20 °C). Lines indicate three-exponential fits. c Kinetics of initial binding of ArfB(540Q) to P + 0(Flu), P + 9(Flu), and P + 30(Flu) complexes. Plotted is the concentration dependence of the apparent rate constant (k_app1) for the predominant association phase in (b). Data represents the mean values of two biological replicates with up to six technical replicates each. The association (k_ON) and dissociation (k_OFF) rate constants are determined by linear fitting of the concentration dependence. Errors of k_ON and k_OFF values are SEM of the fit. d Kinetics of peptidyl-tRNA hydrolysis. Time courses of hydrolysis with P + 0 ribosome complex (0.15 µM) and ArfB (0.2–2 µM) (37 °C). Data represented as mean values of two biological replicates. e Comparison of peptidyl-tRNA hydrolysis rates on P + 0 and P + 9 complexes. Values are obtained by exponential fitting of the rapid phase of the hydrolysis time courses (d and Supplementary Fig. 4f). Error bars represent the SEM of the fit. f Dissociation of ArfB from P + 0, P + 9, and P + 30 complexes. The release of ArfB_GAQ(540Q) (0.1 µM) from the pre-hydrolysis complexes P + 0(Flu), P + 9(Flu), and P + 30(Flu) (0.015 µM) was initiated by rapid mixing with unlabeled P + 0 complexes (1 µM) (20 °C).