1. Volume 27, Issue 2, Pages 250-261 (July 2007)
Progression of the Ribosome Recycling Factor through the Ribosome Dissociates the Two Ribosomal Subunits 
Chandana Barat, Partha P. Datta, V. Samuel Raj, Manjuli R. Sharma, Hideko Kaji, Akira Kaji, Rajendra K. Agrawal 
Molecular Cell 
Volume 27, Issue 2, Pages (July 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1
Stereo Representations of the RRF-Binding Positions on the 70S Ribosome and on the Dissociated 50S Ribosomal Subunit
(A) RRF (red) in position 1 on the 70S ribosome map (yellow, 30S subunit; blue, 50S subunit); and (B) in position 2 on the dissociated 50S subunit map. Two orientations of RRF's domain II within position 1 are indicated. Orientations of the 70S ribosome, shown in the thumbnails to the lower left of each panel, were chosen to optimally reveal the L-shaped feature of RRF densities in each position. The 30S subunit in the thumbnail for (B) is shown as a semitransparent yellow mass, to indicate that the position 2 RRF is observed exclusively on the dissociated 50S ribosomal subunit. Landmarks: I and II, domains I and II, respectively, of RRF. Landmarks of the 30S subunit: hd, head; sp, spur. Landmarks of the 50S subunit: L1, L1 protein; CP, central protuberance; St, L7/L12 stalk; Sb, stalk base; H38 and H69, 23S rRNA helices 38 and 69, respectively.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2
Side-by-Side Comparison of the Two Binding Positions of RRF on the Ribosome, and Binding-Associated Conformational Changes in the Ribosomal Bridge Regions
(A) RRF density (red) on the 50S subunit (blue) portion of the 70S ribosome. A deacylated tRNA (orange) is present in the P/E state. A local conformational change, apparently induced via a direct interaction between the tip of domain II of RRF and the stalk base (Sb) region of the 50S subunit, is highlighted as a dark blue mass (#).
(B) RRF density on the dissociated 50S ribosomal subunit. A weak mass of density (x) observed between RRF and L1 protuberance could be related to an E site tRNA, present in an extremely small fraction of the dissociated 50S subunit population.
(C and D) The density features (solid blue) corresponding to conformational changes in the 50S subunit portion of the 70S•RRF complex map (C), and in the map of the dissociated 50S•RRF complex (D), are superimposed onto the respective 50S maps (semitransparent blue). Locations of intersubunit bridges, B1a, B1b, B2a, B3, and B5, are marked by open ovals. A small mass indicated by an asterisk (∗ in [D]) corresponds to a partial shift of the central protuberance. The 30S ribosomal subunit has been computationally removed from the 70S structure shown in (A), to reveal the RRF and tRNA masses, and in (C), to reveal bridge-related conformational changes. The thumbnail at the bottom, between (C) and (D), depicts the orientation of the ribosome in all four panels. Landmarks: H71, 23S rRNA helix 71; all other landmarks are the same as in Figure 1.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3
Relationship between Position 2 RRF and the P Site tRNA, and Fittings of the Atomic Structure of RRF into the Corresponding EM Densities
(A) The resolution-matched cryo-EM densities of RRF (red) at position 2 and the P site tRNA (semitransparent green; adapted from Gabashvili et al. [2000]) are superimposed.
(B) Transparencies of the two cryo-EM densities have been flipped, to better reveal the relative positions of the two ligands.
(C and D) Atomic structure of the E. coli RRF (PDB ID 1EK8; pink ribbons), docked into the RRF cryo-EM envelope (semitransparent red) in position 1 (C) and in position 2 (D), shown in views that reveal the relative positions of the labeled Cys16 residue (purple balls) and the UG mass (golden yellow, also see Figure S7) within domain I. Note that the fitting shown in (C) corresponds to the position 1 RRF density, representing the predominant orientation of domain II (the IIa configuration, rather than the IIb configuration; see Figures 1A and 2A) in this study. Landmarks: I and II, domains I and II, respectively, of RRF; AC and CCA, anticodon and CCA ends, respectively, of the P site tRNA; landmarks in the thumbnail are the same as in Figure 1.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4
Comparison between the Position 2 Binding of RRF and Positions of the P/P and P/E Site tRNAs, and Conformational Changes in RRF on the Ribosome
(A) Stereo view of atomic structure of RRF (red, domain I; purple, domain II) in position 2 is shown together with mutually exclusive P/P (green, Gabashvili et al., 2000) and P/E (orange) state tRNAs; the latter was derived by docking of an atomic structure of tRNAPhe into the corresponding cryo-EM density (orange mass in Figure 2A). The relative orientations of the two domains of RRF, as derived by docking the atomic structure of the E. coli RRF into the cryo-EM densities corresponding to RRF in both IIb configuration (position 1) and in position 2, matched closely with that in the atomic structure of the Thermotoga maritima RRF (PDB ID 1DD5). Therefore, we also docked the T. maritima structure, as a single rigid body, in the matching positions, and used it to represent the IIb configuration (position 1) and position 2 RRF in this and subsequent figures.
(B) Stereo view, showing superimposed structures of RRF in position 1 (IIa configuration, golden yellow), and position 2 (red). It should be noted that the relative orientation of two RRF domains in its IIb configuration (position 1) is similar to that in position 2.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. Figure 5 The Ribosomal Neighborhood of RRF Binding
Relevant components of the X-ray crystallographic structures of the E. coli (Schuwirth et al., 2005) and T. thermophilus (Yusupov et al., 2001; for the interpretations of the regions that were disordered in E. coli structure) 70S ribosomes, docked into the cryo-EM map of the RRF-bound ribosome, together with RRF (red), are shown in stereo views. (A) RRF in position 1 (IIa configuration), (B) RRF in position 1 (IIb configuration), and (C) RRF in position 2 (IIb configuration). Thumbnail to the lower left depicts the orientation of the ribosome in all three panels. Landmarks: numbers prefixed “L” identify proteins and numbers prefixed “H” identify the 23S rRNA helices of the 50S subunit; P1 and P2, positions 1 and 2 of RRF.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

7. Figure 6
Demonstration of the Steric Clash between the Position 2 RRF and the 30S Subunit
(A) RRF in position 1 (IIb configuration) has been superimposed on the 70S ribosome portion of the PoTC•RRF complex map.
(B) RRF in position 2 has been superimposed on the 50S subunit portion of the 50S•RRF complex. RRF densities (red) were derived by filtering the docked RRF atomic structures to the resolutions of the cryo-EM maps. For RRF to attain its position 2, the 30S subunit must be separated from the 50S subunit by ∼12 Å. All landmarks and color codes are the same as in Figure 1.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

8. Figure 7 Proposed Model of the Ribosome Recycling
(A) RRF (red) on the PoTC in position 1, with its domain II oriented (IIa configuration) toward the stalk base (Sb) of the 50S subunit (semitransparent blue). (B) RRF (still in position 1) with its domain II reoriented (to IIb configuration; see text) toward the 30S subunit (semitransparent yellow). RRF moves to position 2 (C) and (D) from this intermediate position. Such a movement of RRF, possibly catalyzed by EF-G (solid blue) (C), can also be induced in vitro, at least transiently, in a significant proportion of the PoTC, by RRF alone, as indicated by the arrow from (B) to (D). The release of mRNA (green) and the release of P/E tRNA (orange) are depicted from (B)–(D), but the sequence of these two releases could not be inferred from our study. However, both the P/E tRNA and the 30S subunit must dissociate from the PoTC during RRF occupation of position 2 (see text and Figures 4A and 6B). Due to its low binding affinity at position 2, RRF readily dissociates from the 50S subunit (E).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions