Volume 18, Issue 6, Pages 663-674 (June 2005) Mechanism for the Disassembly of the Posttermination Complex Inferred from Cryo-EM Studies  Ning Gao, Andrey V. Zavialov, Wen Li, Jayati Sengupta, Mikel Valle, Richard P. Gursky, Måns Ehrenberg, Joachim Frank  Molecular Cell  Volume 18, Issue 6, Pages 663-674 (June 2005) DOI: 10.1016/j.molcel.2005.05.005 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Overviews of Posttermination Complex in Preratcheting and Postratcheting States Posttermination complex in preratcheting state (A) with P- (dark green) and E-site (golden) tRNAs, and posttermination complex in postratcheting state (B) with P/E-site tRNA (light green) and RRF (purple). (C) and (D) are displayed in transparency to show the orientations of the tRNAs. Landmarks of the 50S subunit: CP, central protuberance; SB, base of the L7/L12 stalk; L1, L1 stalk. Landmarks of the 30S subunit: hd, head; bk, beak; sp, spur. Directions of L1 stalk movement and 30S ratchet movement are indicated by arrows. Molecular Cell 2005 18, 663-674DOI: (10.1016/j.molcel.2005.05.005) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Conformational Changes of the 50S and 30S Subunits upon the Ratchet Movement (A) Superimposition of two aligned 50S subunits from the pre- (transparent red) and postratcheting state (solid blue). (B) Relative orientation of RRF with respect to the hybrid P/E-site tRNA. (C) Relative orientation of RRF with respect to the P- and E-site tRNAs, showing that RRF has an overlapping binding site with the CCA end of P-site tRNA. (D) Superimposition of the 30S subunits (shown from the solvent side) in pre- (transparent red) and postratcheting (solid yellow) positions. (E) As in (D), viewed from the intersubunit side. The dashed oval indicates the region involved in intersubunit bridges with the central protuberance (CP) of the 50S subunit. The dashed circle shows the binding site of RRF on the 30S subunit, which is at the position of protein S12. The dashed peanut–shaped curve represents the upper portion of helix 44, which accounts for two central intersubunit bridges B2a (h44:H69) and B3 (h44:H71). The orientations of the 50S subunits and 70S ribosomes are shown as thumbnails on the left. Molecular Cell 2005 18, 663-674DOI: (10.1016/j.molcel.2005.05.005) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Interactions of RRF with the Ribosome in the Postratcheting State Stereo view of RRF with its surrounding components from the 50S subunit (A), and from the 30S subunit (B). DI and DII represent domain I (tail) and domain II (head) of RRF. The overall orientations of the ribosomal components are shown as thumbnails on the left. Molecular Cell 2005 18, 663-674DOI: (10.1016/j.molcel.2005.05.005) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Overviews of the Cryo-EM Maps of Four 50S Complexes and Conformational Changes of Different Regions in the 50S Subunit (A) Vacant 50S subunit (12.2 Å). (B) 50S + EF-G•GDP•fusidic acid (15.6 Å). (C) 50S + EF-G GDPNP RRF (15.5 Å). (D) 50S subunit from the RRF bound 70S posttermination complex (14.1 Å). (E) The closeup panels 1–4, 5–8, and 9–12 are presented in the same order as in (A)–(D). (F and G) The relative orientation of RRF (dark blue) with EF-G (red) is shown in two different views. Domains of EF-G are labeled as I–V. Arc denotes the arc-like connection between EF-G and L7/L12 stalk of the 50S subunit. Note: (E)6 and (E)7 are displayed with a threshold about 3-fold higher than (E)5, and the arc-like connections of panels (B) and (C) are not continuous under the current threshold, due to the application of a low-pass filter. Molecular Cell 2005 18, 663-674DOI: (10.1016/j.molcel.2005.05.005) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Interaction of RRF with EF-G and Its Implication for the Disassembly of the Posttermination Complex (A) The overall interface of RRF in its 50S conformation (dark blue) with EF-G (red), between the surface of EF-G domain III and IV, and the head domain and part of the hinges of RRF. (B) Superimposition of RRF in its 70S conformation (purple) with GDPNP-state EF-G (red), showing that RRF in its 70S conformation has an overlapping binding site with EF-G•GDPNP. (C) Superimposition of RRF in its 50S conformation (dark blue) with the intersubunit bridges B2a (h44:H69) and B3 (h44:H71), showing the overlapping of RRF head domain (50S conformation) with the h44 and S12. Molecular Cell 2005 18, 663-674DOI: (10.1016/j.molcel.2005.05.005) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Comparison of Two RRF Conformations on the 70S Posttermination Complex Superimposition of RRF in its 50S conformation (dark blue) with RRF in its 70S conformation (purple) on the 50S subunit from the RRF bound 70S posttermination complex (A) and on the 30S subunit from the RRF bound 70S posttermination complex (C). Stereo views of superimposition of the two RRF conformations displayed in docked ribbon structures on the 50S (B) and the 30S subunit (D), respectively. The overall orientations are shown as thumbnails on the left. Molecular Cell 2005 18, 663-674DOI: (10.1016/j.molcel.2005.05.005) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Model for the Disassembly of the Posttermination Complex (A and B) The interconversion of the posttermination complex between preratcheting state (A) and postratcheting state (B) are shown in the framed box. Directions of L1 stalk movement and 30S subunit ratchet movement are indicated by arrows. (C) Posttermination complex (postratcheting state) bound with RRF (70S conformation, purple). (D and E) Posttermination complex bound with both RRF (50S conformation, dark blue) and GTP state EFG (red), viewed in two different orientations. (F and G) Separated 50S and 30S subunits. The star denotes GTP-state EF-G. See text for details. Molecular Cell 2005 18, 663-674DOI: (10.1016/j.molcel.2005.05.005) Copyright © 2005 Elsevier Inc. Terms and Conditions