Decision letter: Cryo-EM visualization of the ribosome in termination complex with apo-RF3 and RF1

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Termination of messenger RNA translation in Bacteria and Archaea is initiated by release factors (RFs) 1 or 2 recognizing a stop codon in the ribosomal A site and releasing the peptide from the P-site transfer RNA. After release, RF-dissociation is facilitated by the G-protein RF3. Structures of ribosomal complexes with RF1 or RF2 alone or with RF3 alone—RF3 bound to a non-hydrolyzable GTP-analog—have been reported. Here, we present the cryo-EM structure of a post-termination ribosome containing both apo-RF3 and RF1. The conformation of RF3 is distinct from those of free RF3•GDP and ribosome-bound RF3•GDP(C/N)P. Furthermore, the conformation of RF1 differs from those observed in RF3-lacking ribosomal complexes. Our study provides structural keys to the mechanism of guanine nucleotide exchange on RF3 and to an L12-mediated ribosomal recruitment of RF3. In conjunction with previous observations, our data provide the foundation to structurally characterize the complete action cycle of the G-protein RF3. https://doi.org/10.7554/eLife.00411.001 eLife digest Ribosomes are complex molecular machines that join amino acids together to form proteins. The order of amino acids in the protein is specified by a strand of messenger RNA (mRNA), and the process of decoding the mRNA into a string of amino acids is called translation. A ribosome consists of two subunits—one large, one small—that come together at a particular site on the mRNA strand called the translation initiation site. The ribosome then moves along the mRNA—joining together amino acids brought to it by transfer RNA (tRNA)—until it reaches a termination site and releases the protein. The ribosome has three sites; the first amino acid to be delivered by a tRNA molecule to the ribosome occupies the site in the middle—also called the P site—and the second amino acid is delivered to the A site. Once the first two amino acids have been joined together, the ribosome moves along the mRNA so that the first amino acid now occupies the third site, called the E or exit site, and the second amino acid occupies the P site, leaving the A site vacant. The third amino acid is then delivered to the A site, and the whole process repeats itself until the ribosome reaches the termination site. Proteins called release factors are responsible for terminating the translation process and releasing the translated string of amino acids, which folds to form a protein. In bacteria this task can by performed by two releases factors, known as RF1 and RF2. However, the release factor must itself be released to leave the ribosome free to translate another strand of mRNA. Pallesen et al. have used cryo-electron microscopy (cryo-EM) to study how a third release factor, RF3, helps to release RF1 from the ribosome in bacteria. In cells, RF3 usually forms a complex with a molecule called GDP, and the cryo-EM studies show that this molecule is released shortly after the RF3•GDP complex enters the ribosome. Once inside the ribosome, RF3 comes into contact with RF1 and with a protein called L12 that is part of the ribosome. A molecule called GTP—which is well known as a source of energy within cells—then binds to RF3, and this causes the shape of the ribosome to change. This change of shape results in the release of RF1 and the formation of a new RF3•GDP complex, which then leaves the ribosome. Further work is needed to fully understand the role of L12 in these events, but a detailed understanding of the mechanism for terminating the translation of mRNA by the ribosome is coming into view. https://doi.org/10.7554/eLife.00411.002 Introduction Termination of bacterial messenger RNA (mRNA) translation depends on recognition of any one of the three stop codons in the ribosomal A site by either class-1 release factor 1 (RF1), recognizing UAA and UAG, or 2 (RF2), recognizing UAA and UGA. RF1 and RF2 induce release of the polypeptide attached to the P-site transfer RNA (tRNA) (for reviews, see Kisselev and Buckingham, 2000; Klaholz, 2011). Subsequent release of the class-1 RF from the post-polypeptide release complex (RC) is facilitated by a class-2 release factor, the G-protein RF3, in a GTP-dependent manner (Freistroffer et al., 1997). RF3, along with initiation factor 2 (IF2), elongation factor Tu (EF-Tu) and elongation factor G (EF-G), constitute a set of four major translational GTPases (Zavialov and Ehrenberg, 2003). Free RF3 has high affinity to GDP; the RF3•GDP complex dissociates very slowly into RF3 and GDP and, thus, free RF3 in the cytoplasm exists mainly in the GDP-form (Zavialov et al., 2001, 2002). In contrast to EF-Tu, for which GDP exchange to GTP is conferred by the guanine nucleotide exchange factor EF-Ts off the ribosome, the guanine nucleotide exchange factor for RF3 is the ribosome in complex with a class-1 RF (Zavialov et al., 2001). GDP-to-GTP exchange occurs rapidly on free IF2 and EF-G in the absence of any exchange factor. As RF3•GDP enters the class-1 RF-bound ribosome, GDP is readily exchanged for GTP, provided the peptide has been hydrolyzed off the P-site tRNA (Zavialov et al., 2002). GTP binding is accompanied by conformational changes in both RF3 and the ribosome (Gao et al., 2007). The change in ribosome structure promotes rapid release of the class-1 RF followed by GTP hydrolysis and, finally, release of RF3 in the GDP form from the ribosome (Zavialov et al., 2001, 2002). Structurally, termination of mRNA translation has been characterized by cryo-EM (Rawat et al., 2006) and X-ray crystal (Petry et al., 2005; Laurberg et al., 2008) structures of A-site bound class-1 RFs interacting with their cognate stop codons. These structures have, in conjunction with molecular dynamics simulations (Sund et al., 2010), contributed to present-day understanding of the principles of stop codon recognition by class-1 RFs and of their activation of the peptidyl transfer center for hydrolysis of the ester bond in peptidyl-tRNA. In addition, the X-ray crystal structure of free RF3•GDP (Gao et al., 2007) and cryo-EM (Gao et al., 2007) and crystal (Jin et al., 2011; Zhou et al., 2012) structures of post-termination and post class-1 RF release ribosomes in complex with RF3 bound to GTP-analogs are known. The latter complexes indicate large structural changes of RF3 upon GTP binding after release of GDP, and suggest those structural changes to drive the ribosome from its macrostate-1 (MS-I; i.e., characterized by lack of intersubunit rotation; for definition of macrostates, see Frank et al., 2007) into a state in which the two ribosomal subunits—30S and 50S—have rotated relative to each other—macrostate-2 (MS-II). Furthermore, these structures of RF3•GDP(N/C)P-bound intersubunit-rotated ribosomes are incompatible with class-1 RF binding (Gao et al., 2007; Jin et al., 2011; Zhou et al., 2012), as previously suggested from biochemical data (Zavialov and Ehrenberg, 2003). In this study we present cryo-EM density maps of the E. coli ribosomal post-peptide-release complex (RC; 70S, tRNAfMet and [Met, stop]-mRNA) in association with RF1 alone and in association with both apo-RF3 and RF1. Complexes were obtained at a residual concentration of free GDP and in the absence of GTP. We show that the latter complex contains a novel form of RF1 in close interaction with the apo-form of RF3 and furthermore infer a direct interaction of RF3 with ribosomal protein L7/L12. We use the present data to provide a structural basis for the mechanism of guanine nucleotide exchange on ribosome-bound RF3 and, in conjunction with previous data, to discuss the whole action cycle of RF3. Results Characterization of the RF3-and-RF1-containing ribosomal release complex We reconstituted ribosomal RCs in vitro by mixing XR7-Met-Stop (UAA) mRNA-programmed E. coli ribosomes containing fMet-tRNAfMet with purified RF1 and apo-RF3 (containing residual guanine nucleotides). To determine the occupancy of RF1 and RF3 in the complex, the reaction was subjected to ultracentrifugation through a sucrose cushion followed by gel analysis in which we estimated the amounts of ribosome-bound RF1 and RF3 by comparing their respective band intensities to band intensities of ribosomal protein S1. The gel showed RF1 and RF3 together on the ribosome in close to 1:1 ratios with S1 in the absence of extra added GDP (Figure 1A,B). When the concentration of extra GDP was increased, the RF3 band decreased gradually and virtually disappeared at 200 μM extra GDP concentration, while the RF1 band remained at 1:1 stoichiometry with S1 (Figure 1A,B). In a second, similar, experiment performed in the absence of RF1, we detected no binding of RF3 to the ribosome—neither to RC nor to 70S—even with no extra added GDP (Figure 1C). From these experiments we could infer that apo-RF3 formed a stable complex with the RF1-bound ribosome at low concentration of free GDP. However, in the absence of RF1 or at elevated GDP concentration, RF3 did not form a stable complex with the ribosome, in line with previous conclusions based on less direct experimental evidence (Zavialov et al., 2001, 2002). Thus, RF3•GDP does not form a stable complex with the RF1-bound ribosome, and we therefore conclude that the reconstituted ribosomal release complex presented in lane 1 of the gel presented in Figure 1A contains the apo-form of RF3 along with RF1. Figure 1 Download asset Open asset Occupancy of RF1 and RF3 in the RC–RF1•RF3 complex and the effect of extra added GDP. (A) Titrating GDP into a release complex (RC) programmed with Met-stop mRNA, and containing fMet-tRNAfMet (in the P site), RF1 and RF3. The intensity of the band corresponding to RF3 decreased gradually to zero with increasing GDP concentration, but the S1 and RF1 intensities remained unaltered. (B) RF3 band intensity relative to S1 and RF1 plotted vs GDP concentration. (C) RC (lanes 1–3) and naked 70S ribosome (lanes 4–6) were incubated with either RF1 alone, RF3 alone or with both RF1 and RF3 as control experiments. RF3 could be detectably bound only to RC—and not to 70S—, when RF1 was present in the complex (lane 3). This confirms that the RF3 bands seen in the gel—in both panels A and C—indeed arise from functional complex formation. https://doi.org/10.7554/eLife.00411.003 Reconstruction of RF1- and apo-RF3/RF1-containing RC The reconstituted RCs were subjected to cryo-EM and single-particle-based reconstruction (Figure 2—figure supplement 1) assisted by image classification. Three classes were identified in total. The first class (∼14,000 projection images; Figure 2—figure supplement 2) yielded a density map of the 70S ribosome in MS-I conformation carrying a P-site tRNA and fragments of an E-site tRNA but with no trace of RF1 or RF3; this class will be disregarded in the following analysis. The second class (∼43,000 projection images; Figure 2A–C) yielded a density map of the 70S ribosome in MS-I conformation carrying RF1 in the A site, a tRNA in the P site and trace density of a tRNA in the E site; the third class (∼29,000 projection images; Figure 2D–F) yielded a density map of the 70S ribosome in MS-I conformation carrying apo-RF3, RF1, a P-site tRNA and trace density of an E-site tRNA. These two maps will be referred to as RC-RF1 and RC-RF1•RF3, respectively. RF1 and RF3 were identified by their shapes and positions on the ribosome (Rawat et al., 2006; Gao et al., 2007, respectively). Figure 2 with 4 supplements see all Download asset Open asset Termination complexes in association with RF1 alone and with both RF1 and apo-RF3. (A) RC-RF1 (resolution 8.4 Å). (B) Close-up of RF1 in the map. (C) RF1 density from (B). Domains 1, 2/4 and 3 of RF1 are labeled. (D) RC–RF1•RF3 (resolution 9.7 Å). (E) Close-up of RF1and apo-RF3 in the map. RF1 contacts both L11—L11 contact point is labeled ‘L11 cp’— and apo-RF3. (F) Density of RF1 and RF3 from (E): RF1 domains labeled in black and RF3 domains 1, 2 and 3 in blue. (G)–(H) In the RC–RF1•RF3 complex, apo-RF3 does not interact with L11 or L6/SRL. Labels: 30S: small ribosomal subunit (yellow), 50S: large ribosomal subunit (blue), cp: central protuberance, L11: ribosomal protein L11, sp: spur, bk: beak, L1: L1 stalk, RF1: release factor 1, RF3: release factor 3, L6/SRL: position of ribosomal protein L6 and the sarcin/ricin loop, L11-NTD: ribosomal protein L11 N-terminal domain, arc: arc-like density. Density maps obtained from the total data set and of the RC-class not occupied by RF1 or RF3 are presented in Figure 2—figure supplement 1 and Figure 2—figure supplement 2, respectively. Additional panels of isolated RF1- and RF1, apo-RF3-densities are presented in Figure 2—figure supplement 3. FSC curves for resolution assessment of all density maps obtained can be found in Figure 2—figure supplement 4. https://doi.org/10.7554/eLife.00411.004 In the RC-RF1 map, domain 3 of RF1 reaches into the peptidyl-transferase center of the 50S ribosomal subunit and the super-domain 2/4 of RF1 occupies the decoding center of the 30S ribosomal subunit. In addition, domain 1 of RF1 contacts the N-terminal domain of protein L11 (L11-NTD) and reaches toward the 30S subunit beak (Figure 2A–C). In the RC-RF1•RF3 map, domain 3 of apo-RF3 contacts domain 1 of RF1 and extends toward the spur of the 30S subunit (Figure 2D–F). Domain 3 of apo-RF3 interacts with 30S protein S12, as observed earlier for RF3 in complex with non-hydrolyzable GTP analogs—GDP(C/N)P—bound to the ribosome in the rotated MS-II form in the absence of a class-1 RF (Gao et al., 2007; Jin et al., 2011; Zhou et al., 2012). However, we observe only a weak interaction between domain 2 of apo-RF3 and 16S rRNA helix 5, whereas the interaction of RF3•GDP(C/N)P with this helix is strong (Gao et al., 2007; Jin et al., 2011; Zhou et al., 2012). Furthermore, we observe domain 1 of RF1 contacting both domain 3 of RF3 and L11-NTD (Figure 2D–F and Figure 2—figure supplement 3B, D). Comparison of the RF1 conformation in our RC-RF1•RF3 map (Figure 2D–F and Figure 2—figure supplement 3B, D) with that in the RC-RF1 map (Figure 2A–C and Figure 2—figure supplement 3A, C) reveals a large conformational change of RF1 in response to the binding of apo-RF3 resulting in the formation of a bridge from apo-RF3 via RF1 to L11 (Figure 2—figure supplement 3). Domain 1 of apo-RF3 is in the vicinity of, but not in contact with, L6/SRL (Figure 2G,H, Figure 3A). In contrast, a direct contact between RF3 and L6/SRL was observed in the post class-1 RF-release RC-RF3•GDP(C/N)P maps (Figure 3B) (Klaholz et al., 2004; Gao et al., 2007; Jin et al., 2011; Zhou et al., 2012), with the ribosome in the rotated MS-II state. Hence, in the apo-RF3 and RF1-containing complex, apo-RF3 is docked to the RC framework only through domain 3, while in the RC-RF3•GDP(C/N)P maps RF3•GDP(C/N)P is seen to be firmly docked to RC through contacts involving domains 1, 2 and 3 (Klaholz et al., 2004; Gao et al., 2007; Jin et al., 2011; Zhou et al., 2012). Figure 3 Download asset Open asset Comparison of L11, L6/SRL, arc (L12-CTD) and apo-RF3 in RC-RF1•RF3 and RC-RF3 maps. (A) Apo-RF3 is observed in contact with L12-CTD (arc) but L12-CTD is not in contact with L11-NTD. Apo-RF3 domain 1 is not in contact with L6/SRL. In contrast (B), RF3•GTP is observed in contact with L6/SRL and in this map (RC-RF3•GTP), L12-CTD is in contact with both RF3 domain 1 and L11-NTD. Both maps are displayed at the resolution of the latter—16 Å—for direct comparison; unsegmented maps were chosen to avoid artifacts on the edges of protein densities and, hence, to present a correct analysis of the interactions occurring. https://doi.org/10.7554/eLife.00411.009 While these observations bear out on the interactions between RF1 and RF3 on the ribosome, an additional finding sheds light on ribosomal recruitment of RF3: Domain 1 of RF3 contains an ‘arc-like’ density (Figure 2G,H, Figure 2—figure supplement 3B, D) clearly separated from the density of the NTD of L11. An ‘arc-like’ density was observed in a similar position, but fully bridging RF3 to L11-NTD in the RC-RF3•GDPNP map (Figure 3B) (Gao et al., 2007). Moreover, a similar bridge to L11-NTD was observed in EF-G- (Agrawal et al., 1998, 1999), EF-Tu- (Stark et al., 1997) and IF2-bound ribosome complexes (Allen et al., 2005). In the case of EF-G, the bridge was subsequently identified as the CTD of L7/L12 (henceforth denoted L12; the difference being an N-terminal acetylation in case of L7) (Datta et al., 2005). The interaction we observe in the present RC-RF1•RF3 map between L12-CTD and apo-RF3 without involvement of L11-NTD has not previously been reported. Interactions involving RF1 and RF3 during termination To interpret the interactions between the RFs and the ribosome in more detail, we used crystal structures of E. coli RF1 (Graille et al., 2005; PDB ID 2B3T) and RF3 (Gao et al., 2007; PDB ID 2H5E) for modeling in our new RC-RF1 and RC-RF1•RF3 maps and the previous RC-RF3•GDPNP map (Gao et al., 2007). Unresolved regions in the structures of both RF1 and RF3 were ab initio- or homology-modeled in accordance with the cryo-EM density maps. We performed molecular dynamics flexible fitting (MDFF) (Trabuco et al., 2009) of our atomic RF1 and RF3 models and the X-ray crystal structure of L12-CTD (Leijonmarck and Liljas, 1987; PDB ID 1CTF) to the above-mentioned maps (Figures 4 and 5). Root-mean-square deviation plots describing changes in the structures as well as changes in the cross-correlation coefficient between structures and density maps over the course of the MDFF fits are presented in Figure 4—figure supplement 1. Figure 4 with 1 supplement see all Download asset Open asset Flexible fitting by MDFF focusing on RF1 conformation and interactions. (A)–(C) Comparison of E. coli RF1 conformations after MDFF. (A) RF1 in RC-RF1. (B) RF1 (and RF3) in RC-RF1•RF3. (C) Superimposition of RF1 from (A) and (B). Arrow in (C) denotes the conformational change of domain 1. (D) In the RC-RF1 complex, RF1 interacts through helices α2 and α3 with the P-rich 310-helix in the L11-NTD. (E) In the RC-RF1•RF3 complex α3 (RF1) interacts with the 310-helix (L11-NTD), while α2 (RF1) interacts with RF3. α2 and α3 denote helices α2 and α3 in domain 1 of RF1; P-rich 310 denotes the P-rich 310 helix in L11-NTD. Root mean square deviation and cross correlation coefficient plots for our MD flexible fitting are presented in Figure 4—figure supplement 1. https://doi.org/10.7554/eLife.00411.010 Figure 5 with 1 supplement see all Download asset Open asset Flexible fitting by MDFF focusing on RF3 conformation and interaction with RF1. (A)–(C) Comparison of E. coli RF3 conformations after MDFF. (A) Crystal structure of free RF3•GDP displayed in its simulated density. (B) apo-RF3 (and RF1) in RC-RF1•RF3 (L12-CTD not displayed) (C) RF3 in RC-RF3•GDPNP (Gao et al., 2007) (L12-CTD not shown). Superimposition (D) of closed conformation RF3•GDP (blue), semi-open apo-RF3 (green) and closed RF3•GTP (gray). Arrows indicate rotations of domain 3. (E) Close-ups of domain 3 conformations from (A)–(D). (F) Candidate residues in domain 3 of RF3 for a charge-based interaction with RF1 and 30S protein S12. (G) Candidate residues in RF1 helix α2 for a charge-based interaction with RF3. RF3 domains labeled in blue; α2 and α3 denote helices α2 and α3 in domain 1 of RF1. Figure 5—figure supplement 1 shows superimposition of apo-RF3 and RF3•GDPNP. https://doi.org/10.7554/eLife.00411.012 In the RC-RF1 map (Figure 2A) we observe RF1 in an open conformation bound to the unrotated, MS-I state ribosome (Figure 4A,C and D), in accordance with the results of earlier studies of this complex (Petry et al., 2005; Rawat et al., 2006; Laurberg et al., 2008). Moreover, our RC-RF1 map conforms to the position of L11 observed in those studies; a position displaced toward the A site when compared to a 70S ribosome with a vacant A site (Laurberg et al., 2008). By comparing RF1 fitted to our RC-RF1 (Figure 4A,D) and RC-RF1•RF3 (Figure 4B,E) maps, the conformational change in domain 1 of RF1 upon RF3 binding can be clearly visualized (Figure 4C). In our RC-RF1 map, helices α2 and α3 in domain 1 are observed in contact with the P-rich 310-helix in L11-NTD (Figure 4A,D) (Petry et al., 2005; Rawat et al., 2006; Laurberg et al., 2008). In the RC-RF1•RF3 map, we observe an RF1-mediated structural bridge between L11-NTD and domain 3 of RF3 (Figure 4E). The stacking between helices α2 and α3 in domain 1 of RF1 is partly disrupted and helix α2 has undergone a rotation by 72° away from L11 (Figure 4C,D and E). Thereby, α2 is positioned within binding distance of domain 3 of RF3 (Figure and while the contact between the of α3 and the P-rich 310-helix in L11 is (Figure (Petry et al., 2005; Laurberg et al., 2008). We compared conformations of apo-RF3 and RF3•GTP fitted to their respective maps with the closed conformation of free RF3•GDP (Figure Apo-RF3 in the RC-RF1•RF3 map is observed in a semi-open conformation characterized by a rotation of its domain 3 away from its domain 1 (Figure RF3•GTP in the RC-RF3 map is observed in its open conformation with domain 3 rotated away from domain 1 when compared to the closed conformation of free RF3•GDP (Figure In the semi-open amino acid and of RF3 form a of or amino acids in two flexible of domain 3 at the with RF1 (Figure the of helix α2 of RF1 in the of interaction with RF3, we 5 amino acids and which are to interaction with RF3 (Figure To light on the of the binding observed between RF1 and apo-RF3 we in the regions their interaction (Figure The of helix α2 in domain 1 of RF1 that contacts RF3 is (Figure whereas the in RF3 is (Figure The observations from fitting (Figure and (Figure suggest that amino acid and of apo-RF3 are in a charge-based interaction with helix α2 of RF1 and residues and of protein (Figure Thus, binding of apo-RF3 to the ribosomal release complex is obtained by formation of a bridge L11-NTD through domain 1 of RF1 with domain 3 of RF3 (Figures and 4E). Figure Download asset Open asset of RF1 and RF3. (A) of RF1. are (B) Close-up of helix α2 in RF1, interaction with RF3 is residues are identified by for a direct interaction with RF3. The of helix α2 is (C) of RF3. (D) Close-up of the two in domain 3 of RF3 or residues for contact with RF1. The of RF3 in this is to a charge-based interaction between RF1 and RF3. In Bacteria RF3 (E) the of RF1 in the α2 of helix α2 by is In this in class-1 RFs from Bacteria and Archaea not RF3 (F) is (G) RF3 from an similar to we observe in E. coli RF3 of the flexible by PDB RF1: RF1: E. coli RF1: from E. coli RF1: RF3: As amino acids in these regions of the two release factors are not we the be (Figure we that in X-ray structures for class-1 RFs from Bacteria coli and Figure the of helix α2 has an In addition, the in RF3 from (Figure et al., 2012) we a similar in the position as we observed for RF3 from E. In contrast, for Bacteria and Archaea RF3 and Figure the of helix α2 is These observations suggest the of charge-based interactions between RF1 and apo-RF3. analysis of the interaction We the interaction between apo-RF3 and RF1 in We RF1 and and compared the of RF3 to these as well as RF1 during translation In our we RCs containing an in the P site and a UAA stop codon in the A site. The RC was in large over RF1, so that of RF1 action were to release from all with earlier (Freistroffer et al., et al., Gao et al., RF3 the of RF1 by a factor of in a GTP-dependent manner (Figure The of RF3 to RF1 and RF1 and was In contrast, compared with of RF1, of the was and of the was the of all RF1 were that the of the and was Figure Download asset Open asset release from RC to of RF1 and release from RC to of RF1 and with of RF3 over The shows for RF1 in the of RF3. In our to of the set of amino acids in our we a set et al., of RF1 from Archaea and the amino acids and we observed as of or and respectively. effect in were not We that with of in our the amino acid of the In light of the of and from the we a in which in the of RF1 is not only in the for interaction with the in RF3 but in a interaction with RF3. We the in RF1 by the to and that to of helix α2 and residues for interaction with RF3. However, a direct interaction between and RF3 be RF3 nucleotide As we observe apo-RF3 in a semi-open the conformation of RF3•GDP has been as closed (Gao et al., 2007) and the conformation of RF3•GTP as open (Gao et al., 2007; Jin et al., 2011; Zhou et al., 2012) (Figure The dynamics in RF3 conformation to the mechanism of nucleotide in the X-ray structure of the of GDP is by interaction with the of RF3 (Figure no is in this (Gao et al., 2007). is part of regions 1 and 2 are known to be flexible regions in activation and of in RF3. We that 1 is in the RF3•GDP crystal structure and therefore not at the position of in semi-open apo-RF3, we observe from the of RF3 (Figure In this is to in nucleotide In the open is observed in a position it is not in of GTP (Figure Moreover, of the of GTP is performed by a the binding of which is by a 1. The is to be for et al., 2012). Hence, the release of GDP from RF3 as it changes conformation from closed to semi-open is by the of from the