Volume 24, Issue 12, Pages 2092-2101 (December 2016) Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy  Ziao Fu, Sandip Kaledhonkar, Anneli Borg, Ming Sun, Bo Chen, Robert A. Grassucci, Måns Ehrenberg, Joachim Frank  Structure  Volume 24, Issue 12, Pages 2092-2101 (December 2016) DOI: 10.1016/j.str.2016.09.014 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 The Recycling Process (A) Simplified scheme for EF-G- and RRF-dependent recycling of the post-termination complex. The 26°C rate constant estimates were obtained and interpolated from experimental estimates of the corresponding rate constants at 20°C and 37°C using the Arrhenius equation (Table S2). [RRF], concentration of RRF; [G], concentration of EF-G; kRRF = 6.6 μM−1 s−1, qRRF = 14 s−1, (kcat/KM)G1 = 23 μM−1 s−1, (kcat)G1 = 15 s−1, (kcat/KM)G2 = 3.8 μM−1 s−1, (kcat)G2 = 4.5 s−1 and kmax = 3.7 s−1. R0, vacant ribosome; R·RRF, RRF-bound ribosome; R·RRF·G, RRF and EF-G-bound ribosome; R·G, EF-G-bound ribosome. Split, the fraction of split ribosomes. (B) Simulation of the recycling reaction at 26°C, starting from the RRF-bound post-termination complex. The distinct ribosome fractions were predicted from the reaction mechanism and its rate constants (A), and plotted as functions of time. The dashed lines indicate the sample composition at 140 ms incubation time. Structure 2016 24, 2092-2101DOI: (10.1016/j.str.2016.09.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Post-termination 70S Ribosome in Complex with RRF and P/E tRNA (A) The PostTC·RRF complex. Gold, 30S subunit; blue, 50S subunit; red, RRF; and orange-red, P/E-site tRNA. h, head; sh, shoulder; sp, spur. (B) Orientation and interaction of RRF and P/E tRNA with the 30S and 50S subunits, respectively. Structure 2016 24, 2092-2101DOI: (10.1016/j.str.2016.09.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Interaction of RRF and tRNA in the PostTC·RRFcontrol and NR-PostTC70S·RRF140 Complexes (A) PostTC·RRF and NR70S·RRF140 complexes in top view. (B) 30S subunit of PostTC·RRF (gold) and NR70S·RRF140 (orange) in solvent and interface views. L1, L1-stalk; h, head; pt, platform; b, body; sp, spur; sh, shoulder; bk, beak. The rotational movement is indicated by dashed lines. Structure 2016 24, 2092-2101DOI: (10.1016/j.str.2016.09.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Segmented Map of the PostTC·RRF·EF-G140 Complex (A) PostTC·RRF·EF-G140 complex. Gold, 30S subunit; blue, 50S subunit; red, RRF; orange-red, P/E tRNA; dark blue, EF-G. (B) Zoom-in views of (A) showing EF-G and RRF interaction. Density is rendered 50% transparent to show the rigid-body fitting of the EF-G and RRF structures. Five EF-G domains and two RRF domains are labeled. Structure 2016 24, 2092-2101DOI: (10.1016/j.str.2016.09.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Movement of Domain II of RRF in PostTC·RRF·EF-G140 Compared with its Position in PostTC·RRF140 The central bridge between 30S and 50S, B2a, involves h44 (yellow) and H69 (blue). S12 protein is shown in green. RRF domain II in orange shown by the fitting of PDB structure (PDB: 1EH1) into its density in the PostTC·RRF·EF-G140 complex. For comparison with the RRF domain II in PostTC·RRF140 (red), the rotational movement of domain II toward bridge B2a is shown with an arrow. The position of domain I (gray) is the same in these two complexes. h, head; sh, shoulder; sp, spur; Sb, L7/L12 stalk base. Thumbnails in the lower left depict the relationship of the views presented here to the view shown in Figure 2. Structure 2016 24, 2092-2101DOI: (10.1016/j.str.2016.09.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 Domain II of RRF Is in Different Positions in PostTC·RRFcontrol, NR-PostTC·RRF140, PostTC·RRF·EF-G140, and 50S·RRF·EF-G140 (A) Comparison of domain II of RRF in PostTC·RRFcontrol and NR-PostTC·RRF140. CP, central protuberance; Sb, stalk base. Helix H69, and domains I and II of RRF are indicated. The position of RRF in PostTC·RRF140 is shown in red. RRF in NR70S·RRF140 is shown in blue mesh. Rotational movement is shown by the dashed lines. (B) Comparison of domain II of RRF in PostTC·RRFcontrol and PostTC·RRF·EF-G140. RRF in PostTC·RRF·EF-G140 is shown as a gray mesh. (C) Comparison of domain II of RRF in PostTC·RRFcontrol and 50S·RRF·EF-G140. RRF in 50S·RRF·EF-G140 is shown as a green mesh. Structure 2016 24, 2092-2101DOI: (10.1016/j.str.2016.09.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 7 30S Subunit Classes (A) Interaction of P/I tRNA with the 30S subunit at 140 ms. Yellow, 30S subunit; green, P-tRNA; orange, P/I tRNA; red, P/E tRNA. The mRNA density is not shown for simplicity. (B) Interaction of IF3 with the 30S subunit at 30 min. h, head; pt, platform; b, body; bk, beak; sh, shoulder; sp, spur. The mRNA density is shown in dark blue. The density attributed to IF3 is segmented out in brown and fitted with the NTD (1TIF, red) and CTD (1TIG, purple) of the IF3 structure. Panels on the left show observed densities, those on the right show the comparison with positions of tRNA (Allen et al., 2005) and IF3 (McCutcheon et al., 1999). Structure 2016 24, 2092-2101DOI: (10.1016/j.str.2016.09.014) Copyright © 2016 Elsevier Ltd Terms and Conditions