Abstract

Article19 February 2004free access Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation Christian MT Spahn Corresponding Author Christian MT Spahn Wadsworth Center, Health Research Inc., Howard Hughes Medical Institute, Albany, NY, USA Wadsworth Center, Albany, NY, USA Institut für Medizinische Physik und Biophysik der Charité, Humboldt Universität zu Berlin, Berlin, Germany Search for more papers by this author Maria G Gomez-Lorenzo Maria G Gomez-Lorenzo Wadsworth Center, Albany, NY, USA Centro Nacional de Biotecnología-CSIC, Campus Universidad Autonoma, Madrid, Spain Search for more papers by this author Robert A Grassucci Robert A Grassucci Wadsworth Center, Health Research Inc., Howard Hughes Medical Institute, Albany, NY, USA Wadsworth Center, Albany, NY, USA Search for more papers by this author Rene Jørgensen Rene Jørgensen Department of Molecular Biology, Aarhus University, Århus, Denmark Search for more papers by this author Gregers R Andersen Gregers R Andersen Department of Molecular Biology, Aarhus University, Århus, Denmark Search for more papers by this author Roland Beckmann Roland Beckmann Institut für Biochemie der Charité, Humboldt Universität zu Berlin, Berlin, Germany Search for more papers by this author Pawel A Penczek Pawel A Penczek University of Texas – Houston Medical School, Houston, TX, USA Search for more papers by this author Juan PG Ballesta Juan PG Ballesta Centro de Biología Molecular ‘Severo Ochoa’, CSIC and UAM de Madrid, Madrid, Spain Search for more papers by this author Joachim Frank Corresponding Author Joachim Frank Wadsworth Center, Health Research Inc., Howard Hughes Medical Institute, Albany, NY, USA Wadsworth Center, Albany, NY, USA Department of Biomedical Science, State University of New York at Albany, USA Search for more papers by this author Christian MT Spahn Corresponding Author Christian MT Spahn Wadsworth Center, Health Research Inc., Howard Hughes Medical Institute, Albany, NY, USA Wadsworth Center, Albany, NY, USA Institut für Medizinische Physik und Biophysik der Charité, Humboldt Universität zu Berlin, Berlin, Germany Search for more papers by this author Maria G Gomez-Lorenzo Maria G Gomez-Lorenzo Wadsworth Center, Albany, NY, USA Centro Nacional de Biotecnología-CSIC, Campus Universidad Autonoma, Madrid, Spain Search for more papers by this author Robert A Grassucci Robert A Grassucci Wadsworth Center, Health Research Inc., Howard Hughes Medical Institute, Albany, NY, USA Wadsworth Center, Albany, NY, USA Search for more papers by this author Rene Jørgensen Rene Jørgensen Department of Molecular Biology, Aarhus University, Århus, Denmark Search for more papers by this author Gregers R Andersen Gregers R Andersen Department of Molecular Biology, Aarhus University, Århus, Denmark Search for more papers by this author Roland Beckmann Roland Beckmann Institut für Biochemie der Charité, Humboldt Universität zu Berlin, Berlin, Germany Search for more papers by this author Pawel A Penczek Pawel A Penczek University of Texas – Houston Medical School, Houston, TX, USA Search for more papers by this author Juan PG Ballesta Juan PG Ballesta Centro de Biología Molecular ‘Severo Ochoa’, CSIC and UAM de Madrid, Madrid, Spain Search for more papers by this author Joachim Frank Corresponding Author Joachim Frank Wadsworth Center, Health Research Inc., Howard Hughes Medical Institute, Albany, NY, USA Wadsworth Center, Albany, NY, USA Department of Biomedical Science, State University of New York at Albany, USA Search for more papers by this author Author Information Christian MT Spahn 1,2,3, Maria G Gomez-Lorenzo2,4, Robert A Grassucci1,2, Rene Jørgensen5, Gregers R Andersen5, Roland Beckmann6, Pawel A Penczek7, Juan PG Ballesta8 and Joachim Frank 1,2,9 1Wadsworth Center, Health Research Inc., Howard Hughes Medical Institute, Albany, NY, USA 2Wadsworth Center, Albany, NY, USA 3Institut für Medizinische Physik und Biophysik der Charité, Humboldt Universität zu Berlin, Berlin, Germany 4Centro Nacional de Biotecnología-CSIC, Campus Universidad Autonoma, Madrid, Spain 5Department of Molecular Biology, Aarhus University, Århus, Denmark 6Institut für Biochemie der Charité, Humboldt Universität zu Berlin, Berlin, Germany 7University of Texas – Houston Medical School, Houston, TX, USA 8Centro de Biología Molecular ‘Severo Ochoa’, CSIC and UAM de Madrid, Madrid, Spain 9Department of Biomedical Science, State University of New York at Albany, USA *Corresponding authors. Wadsworth Center, Health Research Inc., Howard Hughes Medical Institute, Empire State Plaza, Albany, NY 12201-0509, USA. Tel.: +1 518 474 7002; Fax: +1 518 486 2191; E-mail: [email protected] für Medizinische Physik und Biophysik, Charité, Ziegelstr. 5-8, 10117 Berlin, Germany. Tel.: +49 30 450524131; Fax: +49 30 450524931; E-mail: [email protected] The EMBO Journal (2004)23:1008-1019https://doi.org/10.1038/sj.emboj.7600102 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info An 11.7-Å-resolution cryo-EM map of the yeast 80S·eEF2 complex in the presence of the antibiotic sordarin was interpreted in molecular terms, revealing large conformational changes within eEF2 and the 80S ribosome, including a rearrangement of the functionally important ribosomal intersubunit bridges. Sordarin positions domain III of eEF2 so that it can interact with the sarcin–ricin loop of 25S rRNA and protein rpS23 (S12p). This particular conformation explains the inhibitory action of sordarin and suggests that eEF2 is stalled on the 80S ribosome in a conformation that has similarities with the GTPase activation state. A ratchet-like subunit rearrangement (RSR) occurs in the 80S·eEF2·sordarin complex that, in contrast to Escherichia coli 70S ribosomes, is also present in vacant 80S ribosomes. A model is suggested, according to which the RSR is part of a mechanism for moving the tRNAs during the translocation reaction. Introduction Proteins in all living cells are synthesized by ribosomes, which are large, RNA-based macromolecular machines (Ban et al, 2000; Wimberly et al, 2000). During protein synthesis, the macromolecular ligands of the ribosome, that is, mRNA, tRNAs, as well as the nascent peptide chain, move through the ribosome in a precise and controlled manner. These movements of the ligands are accompanied and facilitated by corresponding movements in the ribosome itself (Spahn and Nierhaus, 1998; Frank and Agrawal, 2000; Noller et al, 2002). In line with the paradigm of the ribosome as a molecular machine, structural investigations of ribosomes in defined functional states by cryo-EM and X-ray crystallography are providing increasing evidence for movements of ribosomal parts and are thereby providing a first direct look into the dynamic behavior of the ribosome (for reviews, see Ramakrishnan and Moore, 2001; Ramakrishnan, 2002; Yonath, 2002). The translocation reaction is accompanied by large-scale movements. During this step, the ribosome changes from the pre-translocational (PRE) to the post-translocational (POST) state as the A- and P-site bound tRNAs move to the P and E sites, respectively. The translocation step is catalyzed by elongation factor EF-G in prokaryotes and eEF2 in eukaryotes. In the classical view of translocation, EF-G/eEF2 acts by a GTPase switch mechanism similar to a regulatory G protein (Kaziro, 1978). However, this view was challenged by recent time-resolved pre-steady-state experiments (Rodnina et al, 1997). These experiments indicated that GTP hydrolysis by EF-G precedes tRNA translocation and that the release of inorganic phosphate is coupled with the tRNA translocation step, suggesting that the chemical energy of GTP hydrolysis is used to perform mechanical work on the ribosome. However, it should be noted that mere binding of EF-G without GTP hydrolysis plays a major role in the catalysis of tRNA translocation and contributes more to the acceleration of translocation than GTP hydrolysis (1000-fold versus 50-fold acceleration, respectively; Rodnina et al, 2001). The molecular mechanism of translocation and its catalysis by EF-G/eEF2 is largely unknown. However, binding of prokaryotic EF-G induces large-scale conformational changes in the ribosome that might be related to tRNA movement (Frank and Agrawal, 2000). Much less information is available about eukaryotic ribosomes, but an earlier cryo-EM structure of eEF2 bound to the yeast 80S ribosome at 17-Å resolution showed overall similarity to the prokaryotic system as well as some significant differences (Gomez-Lorenzo et al, 2000). We present here a highly improved cryo-EM map of the yeast eEF2·80S complex in the presence of the antibiotic sordarin at 11.7-Å resolution. Docking of the recent eEF2·sordarin X-ray structure (Jørgensen et al, 2003) and of a molecular model for the yeast 80S ribosome (Beckmann et al, 2001; Spahn et al, 2001a) reveals large conformational changes within eEF2 and the 80S ribosome. Interpreted in molecular terms, these indicate how the ribosome might actively facilitate tRNA translocation. In addition, the mechanism for the inhibitory action of the antifungal drug sordarin is suggested. Results and discussion Cryo-EM reconstruction of the ribosomal eEF2·80S complex and docking of atomic models The reconstruction of the 80S·eEF2·sordarin complex (Figure 1) at 11.7-Å resolution (0.5 threshold; Supplementary Figure S1) was interpreted in molecular terms by docking the recently determined eEF2·sordarin (eEF2-sor) X-ray structure (Jørgensen et al, 2003) and atomic models of ribosomal components into the density map. The docking of ribosomal component models was facilitated by a previous atomic model of the POST 80S ribosome based on a 15.4-Å cryo-EM map (Spahn et al, 2001a). For this purpose, the cryo-EM map of the 80S·eEF2·sordarin complex was brought into the same orientation as the map of the POST 80S ribosome (Spahn et al, 2001a) by first aligning the 60S subunit parts of both maps. After this alignment, it became obvious that the 40S subunits of the two maps differ in position and conformation (see below). Therefore, a crosscorrelation-based 3D orientation search was performed (Spahn et al, 2001b) to find the alignment parameters for the head and body/platform domains of the 40S part of the 80S·eEF2·sordarin map relative to the map of the POST 80S ribosome (Table I). Different alignment parameters for body/platform domains and the head domain were obtained and subsequently used to transform the corresponding parts of our previous atomic model of the translating 80S ribosome (Spahn et al, 2001a). The fitting was further improved by moving nonfitting parts (e.g., RNA helices) as rigid bodies relative to the rest of the model in order to account for local conformational changes in the 40S and 60S subunits. It is estimated that atomic models of known substructures can be positioned in a cryo-EM map with an accuracy exceeding the resolution of the map several-fold (Rossmann, 2000). Indeed, estimation of the accuracy of docking for eEF2 by correlation traces (Valle et al, 2003b) indicated a positional accuracy of better than ±2 Å (Supplementary Figure S2). The resulting atomic model allows an interpretation of the interaction between eEF2 and the 80S ribosome and an analysis of conformational changes within the yeast 80S ribosome at the molecular level. Figure 1.A 11.7-Å-resolution cryo-EM map of the yeast 80S·eEF2·sordarin complex. The cryo-EM map is shown (A) from the side; (B) from the top ; (C) from the 60S side, with 60S removed; and (D) from the 40S side, with 40S removed. The ribosomal 40S subunit is painted yellow, the 60S subunit blue and eEF2 red. Landmarks for the 40S subunit: b, body; bk, beak; h, head; lf, left foot; rf, right foot; pt, platform; sh, shoulder; sp, spur. Landmarks for the 60S subunit: CP, central protuberance; L1, L1 protuberance; SB, stalk base; St, stalk; H34, helix 34; H38, helix 38; SRL, sarcin–ricin loop. Download figure Download PowerPoint Table 1. Orientation search and alignment between different parts of the 80S ribosome Initial alignment at 60S subunits Alignment at complete 40S subunit Alignment at 40S body/platform Alignment at 40S head Angles: Angles: Angles: Angles: α=0° α=5° α=5° α=4° β=0° β=−4° β=−3° β=−14° γ=0° γ=1° γ=1° γ=7° Crosscorrelation coefficientAngles α/β/γ (in case of optimal alignment) 60S subunit 0.89 0.76 n.d. n.d. 0/0/0 40S subunit body/platform 0.83 0.92 0.93 0.59 5/−3/1 40S subunit head 0.74 0.82 0.80 0.92 4/−14/7 The corresponding ribosomal parts of the POST 80S ribosome (Spahn et al, 2001a) and of the 80S·eEF2·sordarin complex are compared in different orientations with the crosscorrelation coefficient. The relative rotational orientation is given in an orthogonal coordinate system, where the x-axis is parallel to the anticodon stem helix of the P-site bound tRNA, the y-axis is parallel to the acceptor stem helix, and the z-axis points from the A-site region toward the E-site region (see Spahn et al, 2001b). The cartoon shows the outline of the 40S subunit from the 60S site and the outline of the P-site bound tRNA, together with the axes representing the coordinate system. The angles α, β and γ describe rotations around the x-, y- and z-axis, respectively. n.d., not determined. Optimal alignment for a certain ribosomal region is indicated by bold numbers and by the angles in the table. The angles describe the rotational rearrangement from the conformation of the POST 80S ribosome to the conformation of the 80S·eEF2·sordarin complex. Large-scale conformational change in eEF2 upon binding to the 80S ribosome The quality of the cryo-EM map allowed eEF2-sor to be docked unambiguously (Figure 2A and B). However, it was necessary to separate the eEF2 structure into two blocks corresponding to domains I/G′/II and III–V and to fit these separately as rigid bodies. The resulting model for ribosome-bound eEF2 (eEF2-cryo) is distinct from both eEF2-sor and apo-eEF2, the nucleotide-free form of eEF2 (Jørgensen et al, 2003). The rotation of domains III–V relative to domains I/G′/II in eEF2-cryo is somewhere intermediate between the two X-ray structures, and closer to that for apo-eEF2 (Figure 2A). Figure 2.Docking of the X-ray model for eEF2 into the cryo-EM density and interactions between eEF2 and the 80S ribosome. Fitting of eEF2 (colored ribbon representation) into the corresponding cryo-EM density (A, B) and ribosomal environment of eEF2 (C, D). Thumbnails are included as an orientation aid. In (A), the X-ray structures of eEF2·sordarin (black, thin ribbon, designated sor) and the apo form of eEF2 (gray, thin ribbon, designated apo) are shown superposed onto domains I and II of the 80S bound eEF2, in order to show the conformational changes. The domains of ribosome-bound eEF2 are color-coded: G (pink), G′ (orange), II (green), III (purple), IV (red), V (cyan). Fitted atomic models of eEF2 and ribosomal components (gray ribbons) in the neighborhood of the factor in ribbon representation are displayed in stereo (C, D). The upper panel (C) focuses on the interactions between the 40S subunit and eEF2, and the lower panel (D) shows a close-up on the stalk base region of the 60S subunit. Residues of eEF2 that possibly interact with the 40S subunit are highlighted in yellow, residues that might be in contact with the 60S subunit are in blue and their numbers are indicated. (C) includes the position of the P-site bound tRNA (in green) that was derived from the POST 80S complex (Spahn et al, 2001a), in order to show the neighborhood of the tip of domain IV of eEF2 to the tRNA anticodon:codon complex. Download figure Download PowerPoint The conformation of apo-eEF2 is thought to be closely related to both the eEF2·GDP and eEF2·GTP forms and should, therefore, represent the conformation of eEF2 in solution (Jørgensen et al, 2003). This leads us to conclude that the conformational change from apo-eEF2 to eEF2-cryo (Figure 2A) occurs upon ribosome binding and that it represents a structural transition that occurs during the translocation reaction. This conformational change is overall similar to the one that has been observed in the Escherichia coli system (Agrawal et al, 1999). However, the conformational change in the yeast system is more pronounced, and the tip of domain IV moves by some 25–30 Å between apo-eEF2 and eEF2-cryo. Additionally, a movement of domain III of eEF2 relative to domains IV and V that is present in eEF2-sor but not in apo-eEF2 (Jørgensen et al, 2003) persists in the structure of eEF2-cryo. The conformational change from apo-eEF2 to eEF2-cryo involves a rotation of domains III, IV and V relative to domains I and II and an additional rearrangement of domain III. Molecular interactions between eEF2 and the 80S ribosome The interaction between eEF2·sordarin and the yeast 80S ribosome involves both ribosomal subunits and all five domains of the factor (Figures 1, 2C and D). The α-sarcin-ricin loop (SRL, H95 of 25S rRNA; we will designate rRNA helices of the large subunit (LSU) by Hmn, where mn is the helix number, and helices of the small subunit by hmn), the so-called GTPase-associated center (GAC; rpL12, H43, H44), and the P proteins (P0, P1α, P1β, P2α, P2β; Ballesta and Remacha, 1996) are contact elements of the large ribosomal subunit (Table II). The latter two elements form the stalk base and stalk, respectively, of the 60S ribosomal subunit. An additional contact element on the 60S ribosomal subunit is the intersubunit bridge B2a. With respect to the 40S subunit, eEF2 interacts with the rRNA (h5, 15, 33, 34, 44) in the head and body domains, as well as the ribosomal protein rpS23 (S12p). Table 2. Contacts between eEF2 and the 80S ribosome from yeast Domain of eEF2 eEF2 positiona Ribosomal subunit rRNA helixb or ribosomal protein rRNA or protein positiona,c, a,c I H27–D29 (G1 motif, P-loop) 60S H95 (SRL) 2656/2663 min I D110 60S H95 (SRL) 2660–2662 I E166 60S rpL9 (L6p) H96, G119 I Q176–T191 60S P proteins II K391 40S h5 359 II R433 40S h15 368 III N499–P502 40S rpS23 (S12p) N99 III L536, E538, E539 60S H95 (SRL) 2660–2662 IV P580–K582 60S H69 1911–1913 lo IV N581, Q704 40S H44 1492, 1493 IV (inset) K613, R617 40S H33 1044 IV Q654–H657 40S H34 1208 V E737, Q738 60S H44 1095 V E757 60S H43 1067 V S745, K749 60S H89 2473 V R760–G762 60S rpL12 (L11p) K24–K29 a Amino acids and nucleotides are given that are closest to the observed contact. b 18S rRNA helices are designated with hmn, where mn is the helix number and 25S rRNA helices with Hmn. rRNA nucleotide numbers are according to E. coli numbering. lo: loop; min: minor groove. The highly conserved SRL of the LSU rRNA is an essential element for the binding and function of several translation factors, forming a strong interaction with the GTP-binding face of domain I (Figure 2D, Table II). The minor-groove side of the SRL stem is adjacent to His27-Asp29 of eEF2. This eEF2 sequence is part of the highly conserved G1 motif, which comprises the phosphate-binding loop (P-loop) (Jørgensen et al, 2003). A second tentative contact involves the tetraloop of the SRL and the G3 motif of eEF2 around Asp110. In addition, the interaction of domain I of eEF2 with the ribosome appears to involve rpL9 (L6p). Interestingly, the position of domain III of eEF2·sordarin appears to be such that the conserved sequence around Leu536 interacts with the tetraloop of the SRL (Figure 2D, Table II). An equivalent interaction between the SRL and domain III of EF-G has not yet been described within the bacterial system. A second ribosomal determinant of translation factor interaction is the GAC in combination with the stalk proteins. These elements undergo a very complex interaction with eEF2 that is to some extent different from the corresponding interaction with EF-G in bacteria (Gomez-Lorenzo et al, 2000). In a separate study, we have identified an interaction between the P proteins and the α-helix D of domain I of eEF2 (Gomez et al, 2004 in preparation). The apical loops of H43 and H44 as well as rpL12 interact with domain V of eEF2 (Figure 2D, Table II). Domain V of eEF2 is further contacted by the apical loop of H89, which from its location could transmit conformational signals into the peptidyltransferase center. The tip of domain IV of eEF2 interacts with both the 40S and 60S subunit, in the region of the decoding center. It contacts the top part of h44 and the apical loop of H69, which form the intersubunit bridge B2a (Figure 2C, Table II). rpS23 (S12p), located adjacent to the decoding center, interacts with domain III of eEF2. As domain III also interacts with the SRL (see above), this contact could be part of an information relay system between the decoding center and the SRL. A prominent difference from EF-G is the prong-like appearance of domain IV of eEF2 (Gomez-Lorenzo et al, 2000), attributed to an insertion in domain IV and a C-terminal addition (Jørgensen et al, 2003). These changes lead to a yeast-specific interaction between domain IV of eEF2 and h33 in the head of the 40S subunit. The 40S subunit also interacts with domain II of eEF2. This interaction with h5 and h15 at the shoulder of the 40S subunit (Figure 2C, Table II) appears to be similar to the corresponding interaction in the E. coli system (Frank and Agrawal, 2001). In line with our previous suggestion (Gomez-Lorenzo et al, 2000), an interaction between the tip of domain IV of eEF2 and the P-site bound tRNA is possible according to the more recent maps, with an experimentally determined model of the P-site bound peptidyl-tRNA (Spahn et al, 2001a) and the model of ribosome-bound eEF2·sordarin. In a superposition of both models, His694-Ile698 of eEF2 proves to be close enough to interact with the codon–anticodon duplex between P-site bound tRNA and mRNA (Figure 2C). Moreover, the adjacent residue His699 is post-translationally modified to diphthamide and ADP ribosylation of diphthamide by bacterial toxins that inactivate the factor. Mutations in eEF2 that prevent diphthamide formation impair factor function (Foley et al, 1995). Taken together, this suggests an additional function for the tip of domain IV of eEF2. It is known that the accuracy of codon–anticodon interaction in the A site is enhanced by A1492 and A1493 of SSU rRNA (E. coli numbering), which interact with the minor groove of the codon–anticodon base pairs (Ogle et al, 2002). These A-minor interactions have to be disrupted for the tRNA to move from the A to the P site. The tip of domain IV of eEF2 could take over the stabilization of codon–anticodon pairing during the transition phase. In this way, eEF2 would ensure that the mRNA follows the movement of the tRNAs, thereby reducing the possibility of frameshifts. A ratchet-like subunit rearrangement takes place in the yeast 80S ribosome The binding of EF-G to the E. coli 70S ribosomes induces a ratchet-like subunit rearrangement (RSR) within the 70S ribosome (Frank and Agrawal, 2000). The RSR presumably corresponds to a transition state of translocation and is accompanied by a movement of a deacylated tRNA in the P site to a P/E hybrid site (Valle et al, 2003b; Zavialov and Ehrenberg, 2003). Previously, we have not detected an RSR-type conformational change upon eEF2 binding, when the 80S·eEF2·sordarin map at 17.5-Å resolution was compared to the map of the vacant 80S ribosome from yeast (Gomez-Lorenzo et al, 2000). However, when we tried to dock the atomic model of the translating yeast 80S ribosome (Spahn et al, 2001a) into the new 80S·eEF2·sordarin map at 11.7-Å resolution, it became obvious that a pronounced RSR is present in the 80S·eEF2·sordarin complex compared to the translating yeast 80S ribosome (Figure 3; Supplementary data: animation, Supplementary Figure S3). Figure 3.Comparison of the yeast 80S ribosome in two different conformations, and positions of the intersubunit bridges. The 80S·eEF2·sordarin complex (A, B) is compared to the POST 80S ribosome (Spahn et al, 2001a) (C, D). The two maps were computationally aligned at their respective 60S subunits (see text). The 40S subunits in yellow (A, C) and the 60S subunits in blue (B, D) are shown from their intersubunit sides. Intersubunit bridges are color-coded. Bridges that are preserved in both structures are painted green, those that are formed by different components are painted red, and those that are specific for one of the conformations are painted pink. The dashed registration lines intersect at bridge b2c, the center of rotation for the RSR. Arrows (B) indicate the inward movements of the L1 protuberance and the stalk region. Download figure Download PowerPoint A comparison with the older maps of the vacant 80S ribosome and the 80S·eEF2·sordarin map at 17.5-Å resolution (Gomez-Lorenzo et al, 2000) shows that the reason why the RSR has not been observed previously is because the RSR state is already present in the vacant yeast 80S ribosome. The translating yeast 80S ribosome contains a peptidyl-tRNA in the P site (Beckmann et al, 2001; Spahn et al, 2001a) and is therefore in the POST state. The subunit organization of the yeast POST 80S ribosome is similar to the translating or vacant bacterial 70S ribosome, consistent with the hypothesis that the peptidyl moiety in the P site locks the prokaryotic ribosome (Valle et al, 2003b; Zavialov and Ehrenberg, 2003). The unexpected presence of the RSR in the vacant yeast ribosome shows that the conformational properties of ribosomes of yeast are different from those of E. coli. The yeast ribosome can stably adopt the RSR conformation without being stabilized by eEF2. Normal-mode analysis is a computational approach to predict and explore global conformational changes of macromolecular assemblies. One of the motions derived from a normal-mode analysis of the X-ray structure of the Thermus thermophilus 70S ribosome (Yusupov et al, 2001) shows striking similarities to the RSR (Tama et al, 2003). The center of the rotational movement predicted by the normal-mode analysis of the bacterial ribosome was found to be h27, the same center as obtained in our experimental analysis of the RSR in the yeast 80S ribosome (see below). Therefore, the mechanical properties of the ribosome that gives rise to the RSR appear to be evolutionary conserved. This, however, leads to the question of why the conformations of the vacant 70S ribosome and the vacant 80S ribosome are different, that is, the latter shows the RSR and the former does not. One possibility is that the two ribosomal conformations exist in an equilibrium, which is shifted toward one state in prokaryotes and toward the other in eukaryotes. In eukaryotic ribosomes, however, there might be a second factor that influences the dynamic behavior. Most of the evolutionary conserved intersubunit bridges, especially the centrally located RNA–RNA bridges, do not have to be broken for the conformation to switch (Figure 3, Table III). However, molecular interactions do have to be temporarily broken and re-formed between some components for the eukaryotic-specific outer bridges (see below), which requires a larger amount of activation energy for the transition to occur. Therefore, the vacant 80S ribosome might be kinetically trapped in the RSR conformation, and the transition between the two conformations might be impeded without proper catalysis. Table 3. Intersubunit bridges Bridge 60S componenta 40S componenta Difference between eEF2·80S and POST 80Sb B1a H38 (886–888) rpS15/S19p (12) Different 40S component H33 (1033) B1b/c rpL11/L5p (91, 92, 170) rpS15/S19p (70) Different 40S component rpL11/L5p (84) rpSx or rpL11/L5p (34,35) rpS15/S19p (N-term) rpS18/S13p 111–118 B2a H69 (1910–1920) H44 (1408/1493) 3.5–4 Å B2b H68 (1847) H24 (784) 2.5 Å 25S (1939–1941) H45 (1515) B2c H67 (1832) H27 (899) 0.5–1 Å H24 (771) B2d H68 (1848/1895) H23 (698–703) 5–5.5 Å B2e rpL2 (136) H23 (712/713) 4 Å rpL43/L37ae (C-term) H22 (671) 3 Å B3 H71 (1948/1960) H44 (1418/1482) 2–3 Å B4 H34 (716) H20 (580/761) 1.5 Å; no interaction between minor groove of 25S H34 and 18S H11 rpS13/S15p B5a rpL23/L14p H44 (1422) 3.5 Å B5b H62 (1689/1704) H44 (1428/1472) 3–3.5 Å B6 rpL23/L14p (132) H14 No bridge B7 rpL24/L24e (47) H44 (1446) 6 Å eB8 H79 (exp) rpSx Different part of rpSx; stronger eB9 rpLx rpSx/h21 (exp) No bridge eB10 H63 (1713/1747) H11 (272) Different ribosome position H101 rpSx eB11 H101 (exp) H9 (187) Local change rpSx eB12 rpL19/L19e (142) n.d. New bridge a Amino acids and nucleotides are given that are closest to the observed contact; rRNA nucleotide numbers are according to E. coli numbering. b If a distance is given, the corresponding bridge appears to be formed by the same components in both states. The distance was inferred geometrically from the rigid body movement of the 40S subunit associated with the RSR. The actual relative movement might be counteracted by local changes. Local conformational changes in the 40S subunit Part of the RSR of the 80S·eEF2·sordarin complex is a rotational movement of the h