Progression of the Ribosome Recycling Factor through the Ribosome Dissociates the Two Ribosomal Subunits

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1 Article Progression of the Ribosome Recycling Factor through the Ribosome Dissociates the Two Ribosomal Subunits Chandana Barat, 1,5,6 Partha P. Datta, 1,5 V. Samuel Raj, 2 Manjuli R. Sharma, 1 Hideko Kaji, 3 Akira Kaji, 2 and Rajendra K. Agrawal 1,4, * 1 Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY , USA 2 Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 3 Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA 4 Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, NY 12201, USA 5 These authors contributed equally to this work. 6 Present address: Department of Biotechnology, St. Xavier s College, 30 Park Street, Kolkata , West Bengal, India. *Correspondence: agrawal@wadsworth.org DOI /j.molcel SUMMARY After the termination step of translation, the posttermination complex (PoTC), composed of the ribosome, mrna, and a deacylated trna, is processed by the concerted action of the ribosome-recycling factor (RRF), elongation factor G (EF-G), and GTP to prepare the ribosome for a fresh round of protein synthesis. However, the sequential steps of dissociation of the ribosomal subunits, and release of mrna and deacylated trna from the PoTC, are unclear. Using three-dimensional cryoelectron microscopy, in conjunction with undecagold-labeled RRF, we show that RRF is capable of spontaneously moving from its initial binding site on the 70S Escherichia coli ribosome to a site exclusively on the large 50S ribosomal subunit. This movement leads to disruption of crucial intersubunit bridges and thereby to the dissociation of the two ribosomal subunits, the central event in ribosome recycling. Results of this study allow us to propose a model of ribosome recycling. INTRODUCTION The process of translation, in which the genetic information encoded in the messenger RNA (mrna) is converted into the amino-acid sequence of a protein, occurs on the ribosome in four steps: initiation, elongation, termination, and ribosome recycling (Kaji et al., 2001; Ramakrishnan, 2002). After the termination step, the mrna and a deacylated transfer RNA (trna) remain associated with the ribosome. These ligands must be released from the ribosome, and the ribosome itself must dissociate into its two subunits during the recycling step, to enable entry into a new round of protein synthesis. Thus, the recycling process involving RRF is an essential step in prokaryotic and eukaryotic organellar translation and involves the disassembly of the posttermination complex (PoTC) (Kaji et al., 2001; Karimi et al., 1999). Atomic structures of RRF from six bacterial species have been determined by X-ray crystallography (Selmer et al., 1999; Toyoda et al., 2000; Kim et al., 2000; Nakano et al., 2003; Saikrishnan et al., 2005) and by NMR spectroscopy (Yoshida et al., 2001). Each of these structures shows that the factor is composed of two domains: domain I, consisting of three long a-helical bundles, and the smaller domain II, which is an a/b motif. Differing orientations of domain II seen in these structures (see Figure S1 in the Supplemental Data available with this article online) have been attributed to interdomain flexibility, which is thought to be necessary for RRF to function on the ribosome (Toyoda et al., 2000; Yoshida et al., 2001). The revelation of an overall similarity, in the dimensions and shape of RRF, to an L-shaped trna molecule prompted the proposal of structural and functional molecular mimicry between the two molecules (Selmer et al., 1999). The binding positions of RRF on the 70S ribosome have been studied by the hydroxyl radical probing method (Lancaster et al., 2002) and three-dimensional (3D) cryoelectron microscopy (cryo-em) (Agrawal et al., 2004; Gao et al., 2005). The binding position of domain I of RRF was also determined by X-ray crystallography (Wilson et al., 2005); in that study the domain I portion of RRF was soaked into a preformed crystal of 50S ribosomal subunits. Results from all of the studies revealed essentially the same overall binding position of RRF on the 70S ribosome. The position of RRF was found to be such that the long arm would block the two trna-binding sites, namely the aminoacyl (A) and peptidyl (P) sites, of the ribosome, but in a direction oblique to that of the long anticodon stem-loop arm of the trna (Lancaster et al., 2002; Agrawal et al., 2004). Previous studies , , July 20, 2007 ª2007 Elsevier Inc.

2 (Agrawal et al., 2004; Wilson et al., 2005; Gao et al., 2005) also revealed that binding of RRF induces a marked conformational change in the 70S ribosome, involving one of the strongest interribosomal subunit bridges, B2a. This finding suggested a direct role of RRF, by which the factor could facilitate the disassembly of the PoTC by severing the intersubunit bridge, on subsequent binding of elongation factor G (EF-G) (Agrawal et al., 2004). Biochemical and genetic studies have shown that an interaction between RRF and EF-G is important in disassembly of the PoTCs (Rao and Varshney, 2001). Furthermore, initiation factor 3 (IF3) has been implicated in the release of the deacylated trna from the dissociated 30S subunit after the concerted actions of RRF and EF-G on the PoTC (Karimi et al., 1999). However, this concept is not fully supported, either by classical (Hirashima and Kaji, 1973) or recent studies (Peske et al., 2005; Zavialov et al., 2005; for a detailed discussion, see Hirokawa et al. [2006]). In the present study, we show that RRF binds in two different binding positions on the ribosome: the first binding position matches closely with previous determinations (Agrawal et al., 2004; Gao et al., 2005; Lancaster et al., 2002; Wilson et al., 2005), while the second binding position overlaps with the binding position of trna in the ribosomal P site. This finding of the second binding position of RRF allows us to outline the possible sequential steps that are involved in the disassembly of the PoTC. RESULTS AND DISCUSSION To determine the binding positions of RRF on the E. coli ribosome, we first labeled E. coli RRF with undecagold (UG), a heavy-metal cluster (core diameter z 8 Å; Nanoprobes, Yaphank, NY); the UG-labeled RRF (RRF-UG) was then bound to the PoTC. A unique cysteine residue present at position 16 (Cys16), within domain I of RRF (Ichikawa and Kaji, 1989; Kim et al., 2000; Figure S2), was labeled using monomaleimido-ug, which forms a covalent linkage with the SH group of the Cys residue (Hainfeld, 1987; Safer, 1999). After ascertaining that the RRF-UG was active in disassembling the model PoTC (see Experimental Procedures and Figure S3), we visualized the position of the UG label by analysis of 3D cryo-em maps (e.g., Kikkawa et al. [2000] and Datta et al. [2005]) of the PoTCRRF-UG complexes. We were able to capture RRF on the ribosome in two different conformational states and in two different binding positions; each position was marked by a strong density for the UG label. In the following sections, we describe our results and discuss how these two RRF positions could be related to the process of ribosome recycling. Localization of RRF on the Ribosome We obtained 3D cryo-em maps (Frank et al., 2000) of the PoTCRRF-UG complex, henceforth referred to as the 70SRRF-UG complex in the rest of the Results and Discussion section. Application of the supervised classification method (Valle et al., 2002) to the 2D cryo-em data set of the 70SRRF-UG complex revealed a partial dissociation among the 70S ribosome population into ribosomal subunits and enabled separation of the images into more homogeneous subsets, based on individual correlations with several reference maps (see Figures S4 and S5). Four distinct classes, and subsequently the corresponding 3D cryo-em maps, were obtained: (1) the empty 70S ribosome; (2) the ratcheted 70S ribosome (Frank and Agrawal, 2000) with an RRF molecule located in a position similar to that observed previously (Figures 1A and 2A; henceforth referred to as position 1 ; also see Agrawal et al. [2004], Gao et al. [2005], and Wilson et al. [2005]); (3) the empty 50S ribosomal subunit; and (4) the 50S subunit with RRF bound in a completely new position, position 2 (Figures 1B and 2B). The new position, found exclusively on the dissociated 50S subunit, appears to be a short-lived state that is attained after the movement of RRF out of position 1 (see below). The density assigned to RRF in position 1 was isolated through comparison between the cryo-em map of the 70SRRF-UG complex and the map of the empty 70S ribosome. RRF in position 1 is seen as a complex density mass, with a long cylindrical arm and two short, bifurcating arms, emerging from the same end of the long arm but having unequal electron densities. The long arm was readily identified as domain I of RRF (Agrawal et al., 2004; Wilson et al., 2005). The two short arms represent two discrete orientations of domain II of RRF on the ribosome. The domain II position represented by the stronger of the two short-arm masses corresponds to the position of domain II suggested from the hydroxyl radical probing study (Lancaster et al., 2002; henceforth referred to as the IIa configuration of RRF in position 1). The domain II position represented by the weaker of the two short-arm masses corresponds to the position of domain II as inferred in the previous cryo-em study (Agrawal et al., 2004; henceforth referred to as the IIb configuration of RRF in position 1; also see Figure S6). The domain IIb configuration has also been proposed in an X-ray crystallographic study (Wilson et al., 2005). (A similar bifurcated density feature, corresponding to two possible orientations of RRF domain II, was observed in the previous cryo-em study [Agrawal et al., 2004]; however, the strengths of the two densities were reversed.) Thus, the complex mass of density in position 1 represents an ensemble average of densities, due to two overlapping binding states of RRF molecules on two separate ribosomes, with the domain I of RRF in two states lying almost in the same position. The density assigned to RRF in position 2 was isolated through comparison between the cryo-em map of the 50SRRF-UG complex and the map of the empty 50S ribosome. This density is located in the ribosomal P site and has a distinct L shape (Figure 1B). The overall length of the long arm of the L matches that of the long arms of both RRF and trna and would almost completely overlap the anticodon stem-loop arm of a trna in the ribosomal P site (Gabashvili et al., 2000; Yusupov et al., 2001; Selmer 27, , July 20, 2007 ª2007 Elsevier Inc. 251

3 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. et al., 2006). However, the anticodon of the P site trna, which interacts with mrna on the 30S ribosomal subunit, would overlap only partially with the outer bend of the elbow of the density corresponding to RRF (Figures 3A and 3B). In addition, there would be no overlap between the CCA arm of the P site trna and the density corresponding to RRF. In fact, the short arm of the L-shaped density is oriented so as to be diagonally opposed to the CCA arm of the P site trna (Figures 3A, 3B, and 4A; cf. Selmer et al. [1999]). Therefore, the mass of density that we see must correspond to RRF in its position 2. This attribution was further verified by presence of a distinct density feature corresponding to the UG label on RRF. Comparison of the maps of UG-labeled complexes with respective unlabeled controls reveals that the overall shape of the labeled region does not change upon incorporation of the UG label. However, the RRF moiety in each of the two positions showed a unique high-density spot (within the long arm [domain I] of the L-shaped RRF density) that persisted after imposition of a very high density threshold and thus was readily attributable to UG (Figures 3C and 3D; also see Figure S7). We further validated the UG positions through calculation of difference maps for both position 1 (using cryo-em maps of the 70SRRF-UG complex and 70SRRF complex; Agrawal et al., 2004) and position 2 (using the cryo-em maps of the 50SRRF-UG complex and the 50S subunit, to which a filtered and resolution-matched X-ray structure of RRF was added in the matching position). Presence of a mass of density corresponding to UG on position 2 RRF was also verified by the subtraction of a cryo-em map of a complex containing a trna bound at the P site of the 50S ribosomal subunit (isolated by computationally removing the 30S portion of the cryo-em map of the Met 70SfMet-tRNA f complex; Gabashvili et al., 2000) from the map of the 50SRRF-UG complex , , July 20, 2007 ª2007 Elsevier Inc.

4 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 70SRRF complex map (C), and in the map of the dissociated 50SRRF 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. In order to exclude the possibility that the binding of RRF to position 2 is a consequence merely of an altered buffer condition (as absence of reducing agents in our buffer was a requirement to maintain the Cys16-UG coupling; see Experimental Procedures) and/or the use of UG labeling, we reanalyzed our earlier data set of the 70SRRF complex (Agrawal et al., 2004) using the same scheme of supervised classification (Figure S4). Indeed, we found partial dissociation of the 70S ribosome in that data set. A cryo-em map computed from the further-classified small fraction of the 50S particle images showed an RRF signature density in the position 2 region (Figure S8). These results further support our observation of the position 2 binding of RRF, and an RRF-dependent partial and spontaneous dissociation of the 70S ribosome. Moreover, our biochemical data (Figure S3B) also show a limited dissociation into ribosomal subunits, upon incubation of 70S ribosomes with RRF alone. Conformational Changes of the Ribosome Due to RRF Binding Comparisons between the cryo-em maps of the RRFbound and unbound ribosomes, obtained under identical buffer conditions, revealed conformational changes in the ribosome that are associated with binding of RRF (Figures 2C and 2D). Most of the conformational changes described in the previous cryo-em study of the 70SRRF complex (Agrawal et al., 2004) have been reproduced in this study. In addition, we here observe marked conformational differences between the 50S subunit portion of the 70S ribosome with position 1 RRF, and the dissociated 50S subunit with position 2 RRF. The L7/L12 stalk (St), which is clearly extended in the 70SRRF map (position 1), is disordered in the map of the dissociated 50SRRF complex (position 2; Figure 2, compare [A] and [C] with [B] and [D], respectively). However, the functional significance of the conformational changes in the stalk region is not yet understood. For both positions of RRF, conformational changes occurred in the vicinity of 23S rrna helices 69 and 71 of the 50S subunit; these helices are involved in the formation of several of the strongest bridges (B2a, B3, and B5; Figures 2C and 2D) between the two ribosomal subunits (Gabashvili et al., 2000; Yusupov et al., 2001; Selmer et al., 2006). We find that a large conformational change in this region is initiated with binding of RRF in position 1 on the 70S ribosome (Figure 2C; also see Agrawal et al. [2004]) and becomes significantly amplified with RRF in position 2 on the dissociated 50S subunit (Figure 2D). Furthermore, the conformations of the 23S rrna helix 38 and the central protuberance (CP) of the 50S subunit change upon RRF binding in position 2. Both components shift toward the position 2 RRF. These two shifts affect the configurations of intersubunit bridges B1a and B1b and could be partially responsible 27, , July 20, 2007 ª2007 Elsevier Inc. 253

5 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. for the above-described dissociation of the 70S ribosome into its two subunits. Because such pronounced conformational changes are not observed in the 50S subunit that is dissociated from the 70S ribosome upon treatment with low-concentration Mg 2+ (<1 mm) (P.P.D. and R.K.A., unpublished data), they must be triggered by the binding of RRF to position 2. In an earlier study of RRF binding to a predissociated 50S ribosome in the presence of EF-G (Gao et al., 2005), it was suggested that domain II of RRF (in a position similar to our position 1) would sterically interfere with association of the 30S subunit. However, in that study, domain II of RRF was found to be sandwiched between EF-G s domain IV (Agrawal et al., 1998) and 23S rrna helices 69/ 71 of the 50S subunit, such that the RRF s movement to position 2 and subsequent release from the ribosome (Kiel et al., 2003) would be precluded. However, that finding corroborated the results of previous study (Kiel et al., 2003), suggesting that RRF bound to the predissociated 50S subunit cannot be released by EF-G, whereas RRF bound to the 70S ribosome is readily released in the presence of EF-G, presumably via position 2 identified in the present study. Moreover, with the predissociated 50S subunit, only a weak density related to RRF in position 1 is seen (see Figure S9), unless RRF is trapped by domain IV of EF-G (e.g., Gao et al. [2005]). This is not surprising, because the dissociation constant of RRF for the predissociated 50S subunit is significantly higher than that for the 70S ribosome (Hirokawa et al., 2002a, 2002b). Because in the present study RRF was allowed to react with its natural substrate, the PoTC, and because RRF binding in position 2 could not be detected on a predissociated 50S ribosomal subunit when incubated with saturating quantities of RRF (60-fold molar excess) (Gao et al., 2005; also see Figure S9), it is likely that position 2 of RRF is achieved only via the factor s initial binding to the 70S ribosome in position 1, rather than representing a direct binding of RRF to a predissociated 50S subunit. We believe that the conformational changes, induced by binding of RRF at position 1, prepare the 50S subunit within the 70S ribosome for the transition of RRF to position 2. The fact that we did not observe position 1 binding of RRF to 50S subunits that were dissociated upon RRF action could be related to an unfavorable conformation of those 50S subunits. However, the possibility that an extremely small fraction of dissociated 50S subunit population binds RRF in position 1, but is not classified as a separate group in our supervised classification scheme, cannot be ruled out. Comparison of the Cryo-EM Maps with the Atomic Structure of RRF The atomic structure of the E. coli RRF (PDB ID 1EK8) was docked as two separate domains into the RRF density in both of its positions, taking into consideration both the constraints of the envelope of the density and the position of the UG mass (Figures 3C and 3D). Because configuration IIb of RRF in position 1 is the same as derived in the previous study (Agrawal et al., 2004), we present here dockings of the RRF atomic structure into cryo-em densities that correspond to the other position 1 configuration, IIa, and to the position 2. The tight packing of the UG mass within the helix bundle and the slight broadening of the density of the RRF domain I at both positions suggest , , July 20, 2007 ª2007 Elsevier Inc.

6 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 trna Phe 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. that RRF undergoes an intradomain conformational rearrangement upon binding to the ribosome. However, we did not introduce any such rearrangement in our dockings. Additionally, given the two overlapping configurations of domain II (IIa and IIb; Figures 1A and 2A), it is conceivable that domain I undergoes a small shift (up to 5 Å) and/or rotation (up to 15 around its long axis) within position 1, as also inferred from our independent fittings corresponding to both configurations of RRF (Figure 5, compare [A] and [B]). Our fittings reveal a large interdomain rearrangement of RRF on the ribosome. In the IIa configuration, domain II forms an obtuse angle (120 ) with the long axis of domain I, while it is roughly perpendicular (93 ) to that axis in the IIb configuration (Agrawal et al., 2004). In position 2, the domain II of RRF maintains a roughly right angle (IIb configuration) with the long axis of domain I (Figures 4A and 4B). These observations, and the fact that the density corresponding to the IIb configuration is significantly weaker than that in the IIa configuration, lead us to suggest that RRF has moved from position 1 to position 2 via the IIb configuration, maintaining the interdomain angle of 93. This movement would produce a significant steric clash with the 30S subunit in the 70S ribosome, such that the 30S subunit would be separated from the 50S subunit by at least 12 Å (Figure 6). Ribosomal Neighborhood of RRF in Its Two Positions In order to determine the binding position of RRF on the ribosome, we docked the X-ray crystallographic structure of the E. coli 70S ribosome (Schuwirth et al., 2005) into our cryo-em maps of the 70S ribosome and the 50S subunit. Position 2 places RRF in a new biochemical environment of the ribosome (Figure 5C), thereby satisfying some of the hydroxyl radical probing (HRP) data (Lancaster et al., 2002) that are inconsistent with the position 1 siting. For example, the HRP cleavage of 23S rrna helix 38 of the 50S subunit with the probe at amino acid residue 10, within domain I of RRF, could be easily attributed to the fraction of the dissociated 50S subunit population having the RRF bound in position 2. The tip of domain II of the RRF in the IIa configuration (position 1) would interact with the base of the 50S subunit stalk, involving the 23S rrna helix 43/44 (Figure 5A), as also suggested by HRP study (Lancaster et al., 2002), while in the IIb configuration the tip is oriented toward helix 18 of the 16S rrna and protein S12 of the 30S subunit (Agrawal et al., 2004). On the basis of the two binding positions of RRF observed in this study, we speculate that 23S rrna helix 80 and ribosomal protein L16 of the 50S subunit (Figure 5) the two ribosomal elements that lie in the vicinity of the tip of domain I (in both positions) act as anchoring points and perhaps shift along with RRF, during the factor s progression from position 1 to position 2. Indeed, a weak density feature within the difference map computed between the map of the 50S subunit with RRF in position 2 and the computationally isolated 50S subunit portion of the map of the 70SRRF complex suggests a conformational change (not shown) near the helix 80/L16 region, coinciding with the tip of the domain I of RRF. 27, , July 20, 2007 ª2007 Elsevier Inc. 255

7 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 , , July 20, 2007 ª2007 Elsevier Inc.

8 mechanism of ribosome recycling. Superimposition of the fitted RRF structure in both of its positions onto the structure of EF-G (Agrawal et al., 1999a; Valle et al., 2003) shows that the RRF in its position 1 (in both configurations IIa and IIb) would suffer serious steric clashes with domains III V of EF-G in both the GTP and GDP states (see Figure S10). In position 2, RRF does not show any steric clash with EF-G. Thus, in the presence of EF-G, RRF can reside in position 2, but not in position 1. In other words, upon EF-G binding to the PoTC containing RRF, RRF must be displaced from position 1. This observation is consistent with the finding that RRF can coexist with EF-G on the ribosome, but only with very weak affinity (Kiel et al., 2003), such that RRF swiftly dissociates from the ribosome (Seo et al., 2004). 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 PoTCRRF complex map. (B) RRF in position 2 has been superimposed on the 50S subunit portion of the 50SRRF 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. Relationships of RRF-Binding Positions with Those of trnas and EF-G on the Ribosome As pointed out in the previous studies (Lancaster et al., 2002; Agrawal et al., 2004), position 1 of RRF overlaps the binding positions of both A and P site trnas, such that the long axes of the two ligands are obliquely oriented. These observations indicated that RRF binding, and trna binding to A and P sites, are mutually exclusive, suggesting that RRF recognizes a ribosome substrate in which both A and P sites are free of trna, as expected in a PoTC, with the 70S ribosome, mrna, and deacylated trna in the P/E state (Agrawal et al., 1999b). In position 2, RRF would partially overlap the anticodon loop of the deacylated trna occupying the ribosomal P/E site (Figure 4A), the site that is found to be occupied in our 70SRRF complex (Figure 1A), but not in the 50SRRF complex. The implication is that the movement of RRF to position 2 has displaced the P/E site trna. Because RRF works in concert with EF-G (Hirashima and Kaji, 1972), knowledge of how the two factors interact on the ribosome is essential for an understanding of the Sequence of Events in the RRF-Mediated Disassembly of the PoTC Based on above observations, we propose a model of disassembly of the PoTC (70S ribosomemrnap/e-trna complex) (Figure 7; also see Movie S1). RRF binds to the PoTC with domain II of RRF facing the stalk base of the 50S subunit (IIa configuration, Figure 7A). In this IIa state, an interaction of the tip of domain II of RRF with the stalk base may trigger the subsequent steps. Because domain II of RRF in this state would be in serious steric conflict with domain V of the incoming EF-G (see Figure S10), the initial interaction of EF-G (see Frank and Agrawal [2001]) might orient the RRF domain II toward the 30S subunit (IIb configuration, Figure 7B). However, our results suggest that the domain II of RRF is capable of spontaneously reorienting to the latter configuration (IIb), given that density attributable to both possible orientations of domain II is observed here and in the previous study (Agrawal et al., 2004). It is most likely that RRF in this intermediate state (IIb configuration in position 1) is ultimately pushed by domain IV of EF-G to position 2 (Figure 7C), without alteration to the IIb configuration of the two domains. Due to disruption of some of the bridges (Figures 2C and 2D), the two ribosomal subunits dissociate, releasing the 30S subunit, as proposed earlier (Hirokawa et al., 2005; Peske et al., 2005; Zavialov et al., 2005). The movement of RRF from position 1 to position 2 (Figures 7B 7D) would ensure the ejection of the deacylated trna from the P/E site, because the anticodon end of the trna would sterically clash with the elbow region of RRF in position 2 (Figure 4A). This series of events is consistent with the results of the kinetic study (Peske et al., 2005) indicating that 63% of releasable trna is released by RRF and EF-G, without addition of IF3, as has also been indicated in other studies (Hirashima and Kaji, 1973; Zavialov et al., 2005). Considering that the separation of the two ribosomal subunits occurs during the RRF progression from position 1 to position 2, and that the deacylated P/E trna is physically displaced by the position 2 RRF, the subunit separation step (Figure 6) would either precede or occur simultaneously with the trna removal step. It is also likely that a partial intersubunit separation, induced during RRF 27, , July 20, 2007 ª2007 Elsevier Inc. 257

9 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). progression from position 1 to position 2, facilitates the mrna removal and allows IF3 to gain access to its binding site on the 30S subunit (Dallas and Noller, 2001), so as to promote the actual subunit dissociation (Singh et al., 2005). The presence of empty 50S subunits suggests (1) that a significant proportion of the 70S ribosomes with bound RRF have dissociated into subunits, presumably during the RRF transition from position 1 to position 2; and (2) that RRF binding in position 2 is very weak and short-lived, given that only 6% of the total selected particles showed RRF in this position (see Experimental Procedures and Figures S4 and S5). However, even during its short lifespan, the occupancy of position 2 RRF leads to changes in conformation of three additional intersubunit bridges (Figure 2D), and it sterically prohibits the 30S subunit from binding to the 50S subunit (Figure 6). Thus, RRF at position 2 could act like a transient antiassociation factor for the dissociated 50S subunit. During the PoTC disassembly, the mrna dissociates from the 30S subunit, which is maintained in its isolated form by binding of IF3 (Hirokawa et al., 2005; Peske et al., 2005), until the next round of translation initiation. In conclusion, the proposed model of recycling best accounts for all of the binding states observed in this study. Our findings suggest that in the absence of EF-G, a nonenzymatic movement of RRF on the ribosome can take place, as had been indirectly inferred from previous studies (Raj et al., 2005; Seo et al., 2004). The transient nature of RRF/EFGGTP-driven subunit dissociation has been recently demonstrated (Hirokawa et al., 2005; Peske et al., 2005; Zavialov et al., 2005). The partial dissociation of the 70S ribosome population observed in the present study could be partly related to the very dilute ribosome concentration, a requirement to obtain an optimum distribution of ribosome particles on the cryo-em grid, and to the presence of large excess of RRF during the grid preparation. Nevertheless, the rapid freezing of the sample on the cryo-em grid has enabled us to capture an otherwise short-lived intermediate. Because only a small population of the 50S subunits had RRF bound at position 2, it is not surprising that detection of such a short-lived state had eluded previous studies. The presence of EF-G would render such an intermediate even more transient. EXPERIMENTAL PROCEDURES Undecagold Labeling of RRF Purified E. coli RRF was incubated for 2 hr at room temperature with a 10-fold molar excess of monomaleimido UG (Nanoprobes, Yaphank, , , July 20, 2007 ª2007 Elsevier Inc.

10 NY), for labeling of its native Cys16 with UG. RRF-UG was chromatographically separated from excess free UG, using a Toyopearl-40 HPLC column in Tris-HCl buffer (ph 6.45). The calculated labeling efficiency based on the relative absorbances of RRF and RRF-UG at 280 nm and 420 nm (see Datta et al. [2005]), and as determined by gel-shift assay (Figure S2), was close to 100%. Binding and Biological Activity of RRF and RRF-UG The model PoTC was obtained as described by Hirokawa and coworkers (Hirokawa et al., 2002b) by treatment of the E. coli polysome preparation with puromycin in a buffer containing 8.2 mm MgSO 4,80 mm NH 4 Cl, and 10 mm Tris (ph 7.4), but without DTT. Binding of RRF- UG to the PoTC was 60% (i.e., 0.6 mol RRF-UG per mol of 70S ribosomes), as assessed by quantitative western blot. However, RRF-UG was efficient in the disassembly of the PoTC (Figure S3A). The PoTC was subjected to sucrose density gradient centrifugation, and the 70S peak was used for RRF-UG binding and cryo-em. The PoTCRRF-UG complex for the cryo-em work was prepared by incubation of the 2 mm PoTC with 20 mm RRF-UG at room temperature in 50 ml buffer, containing 50 mm Tris-HCl (ph 7.5), 10 mm Mg(OAc) 2, 5.5 mm NH 4 Cl, and 25 mm KCl. Dissociation of the 70S ribosomes into subunits was measured by the light scattering method (Figure S3B) as described previously (Hirokawa et al., 2005). Cryo-EM and Image Reconstruction of the PoTCRRF-UG Complex Before dilution of the PoTCRRF-UG complex (to a final concentration of 32 pmol/ml) for cryo-em grid preparation (Wagenknecht et al., 1988), 50-fold molar excess of RRF-UG was added to the buffer (50 mm Tris-HCl [ph 7.5], 10 mm Mg(OAc) 2, 5.5 mm NH 4 Cl, and 25 mm KCl). EM data were collected on a Philips FEI Tecnai F20 field emission gun electron microscope, equipped with a low-dose kit and an Oxford cryotransfer holder, at a magnification of 50,7603. The majority of the data were collected at close-to-focus settings (underfocus setting ranging from 0.7 to 1.8 mm), so as to enhance the signal due to the strongly scattering UG label on the ribonucleoprotein background (also see Datta et al. [2005]). A total of 109 micrographs for the PoTCRRF-UG complex were scanned on a Zeiss flatbed scanner, with a step size of 14 mm, corresponding to 2.76 Å on the object scale. Initially, 136,234 images were picked and sorted into 18 defocus groups (ranging from 0.68 to 3.10 mm underfocus). The projection matching procedure (Penczek et al., 1994) of the SPIDER software (Frank et al., 2000) was used to obtain CTFcorrected (Penczek et al., 1997) 3D cryo-em maps. Supervised Classification of the 2D Cryo-EM Data Set Initial reconstruction using a manually selected subset of 98,238 images indicated significant heterogeneity in the data set. Therefore, the data set was subjected to supervised classification (Valle et al., 2002). The detailed procedure is provided in the Supplemental Experimental Procedures, where the scheme of supervised classification and the related data are presented in Figures S4 and S5, respectively. Through classification, we obtained four distinct classes: (1) empty 70S ribosomes (represented by 18,288 images, 19% of the total selected images), (2) RRF-bound ratcheted 70S ribosome (represented by 36,439 images, 37% of the total images), (3) empty 50S subunits (37,803 images, 38% of the total images), and (4) RRF-bound 50S subunits (5708 images, 6% of the total images). The resolutions of the 3D maps of the 70SRRF-UG and 50SRRF-UG complexes were 15.3 Å and 18.4 Å, respectively, according to the FSC criterion with 0.5 cutoff (see Bottcher et al. [1997] and Malhotra et al. [1998]), or 11.0 Å and 15.0 Å, respectively, according to the 3s criterion (Orlova et al., 1997). We filtered the maps according to the 0.5 cutoff criterion. Docking of Atomic Coordinates into the Cryo-EM Maps To optimally incorporate the X-ray crystallographic structure of the RRF (Kim et al., 2000; PDB ID 1EK8), we docked the RRF structure, divided into two domains, which were treated as two separate rigid bodies, into our cryo-em maps of ribosome-rrf complexes, using O(Jones et al., 1991). During our dockings, both the cryo-em envelope and the positional constraints imposed by the UG density were taken into consideration, and the distance between residue Cys16 of RRF and the position of the UG density was held within 16 Å, to accommodate the length of the linker and the radius of the organic shell that surrounds the UG core (Safer, 1999). All resulting coordinates of RRF were energy minimized, to relieve strain from any unfavorable steric interactions. Insight II (Accelrys, USA), with the molecular mechanics/dynamics program DISCOVER, was used to perform energy minimization. The final distances between the sulfur atom of the target Cys residue and the center of the mass corresponding to UG density were between 8 and 10 Å, slightly shorter than expected (Safer 1999; Datta et al., 2005). In order to interpret the ribosomal neighborhood of RRF in both its positions, we docked the X-ray crystallographic structures of the E. coli (Schuwirth et al., 2005) and T. thermophilus (Yusupov et al., 2001) 70S ribosome as a rigid body into the cryo-em map. The crosscorrelation coefficient (CCC) values between the fitted X-ray coordinates and the corresponding cryo-em density maps were determined after conversion of the fitted coordinates to the density map, through computation of averaged densities within volume elements scale matched to those of the cryo-em map (i.e., a pixel size of 2.76 Å, and after filtration of the X-ray map to the resolution of the cryo-em density map). The CCC values between the X-ray structures and cryo-em densities corresponding to position 1 and position 2 RRFs were in the range. Visualization of the fitted atomic structures and the cryo-em density maps was done with Ribbons (Carson, 1991) and IRIS EXPLORER (Numerical Algorithms group, Downers Grove, IL), respectively. Supplemental Data Supplemental Data include ten figures, one movie, Supplemental Experimental Procedures, and Supplemental References and can be found with this article online at full/27/2/250/dc1/. ACKNOWLEDGMENTS The authors thank T. Booth for help with some of the EM data collection, M. Breedlove for help with image processing, and Drs. Adriana Verschoor and Michael Koonce for critical readings of the manuscript. We thank Dr. Go Hirokawa for critical reading of the manuscript and for allowing us to include his unpublished light-scattering data on subunit dissociation as part of the Supplemental Data (Figure S3B). This work was supported by NIH R01 grants GM61576 (to R.K.A) and GM60429 (to A.K.), and by Creative Biomedical Institute support (to A.K.) and Nippon Paint grant (to H.K.). 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