Fig. 6. SufB2 interferes with elongation complex dynamics during late steps of translocation.

a–c Cartoon representation of elongation as a G37-state SufB2- or ProL-TC is delivered to the A site of a bL9(Cy3)- and uL1(Cy5)-labeled 70 S IC; a in the absence, or b in the presence of EF-G, or c upon using puromycin (Pmn) to deacylate the P site-bound G37-state SufB2 or ProL and generate the corresponding PRE^–A complex. The 30S and 50S subunits are tan and light blue, respectively; the L1 stalk is dark blue; Cy3 and Cy5 are bright green and red spheres, respectively; EF-Tu is pink; EF-G is purple; fMet-tRNA^fMet is dark green; and SufB2 or ProL is dark red. d, e Hypothetical (top) and representative experimentally observed (bottom) E_FRET vs. time trajectories recorded as ProL-TC is delivered to a 70S IC, d in the absence and e in the presence of EF-G as depicted in (a, b). The waiting times associated with k_{70S IC→GS2}, k_GS1→GS2, k_GS2→GS1, and k_GS2→POST are indicated in each hypothetical trajectory. f–h Surface contour plots of the time evolution of population FRET obtained by superimposing individual E_FRET vs. time trajectories in the experiments in (a–c), respectively, for SufB2 (top) and ProL (bottom). N: the number of trajectories used to construct each contour plot. Surface contours are colored as denoted in the population color bars. For pre-steady-state experiments, the black dashed lines indicate the time at which the TC was delivered and the gray shaded areas denote the time required for the majority (54–68%) of the 70S ICs to transition to GS2. Note that the rate of deacylated SufB2 dissociation from the A site under our conditions is similar to that of EF-G-catalyzed translocation, thereby resulting in the buildup of a PRE complex sub-population over 3–20 min post-delivery that lacks an A site tRNA and is incapable of translocation. This sub-population exhibits k_GS1→GS2, k_GS2→GS1, and K_eq values similar to those observed in experiments recorded in the absence of EF-G (Supplementary Table 6).