GTPase activation of elongation factor EF-Tu by the ribosome during decoding
2009; Springer Nature; Volume: 28; Issue: 6 Linguagem: Inglês
10.1038/emboj.2009.26
ISSN1460-2075
AutoresJan-Christian Schuette, F.V. Murphy, Ann C. Kelley, John R. Weir, Jan Giesebrecht, Sean R. Connell, Justus Loerke, Thorsten Mielke, Wei Zhang, Pawel A. Penczek, V. Ramakrishnan, Christian M. T. Spahn,
Tópico(s)Plant Virus Research Studies
ResumoArticle19 February 2009free access GTPase activation of elongation factor EF-Tu by the ribosome during decoding Jan-Christian Schuette Jan-Christian Schuette Institut für Medizinische Physik und Biophysik, Charite-Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Frank V Murphy IV Frank V Murphy IV Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Ann C Kelley Ann C Kelley Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author John R Weir John R Weir Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Jan Giesebrecht Jan Giesebrecht Institut für Medizinische Physik und Biophysik, Charite-Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Sean R Connell Sean R Connell Institut für Medizinische Physik und Biophysik, Charite-Universitätsmedizin Berlin, Berlin, GermanyPresent address: Institut für Organische Chemie, JW Goethe Universität, Max-von-Laue Strasse 7, 60438 Frankfurt am Main, Germany Search for more papers by this author Justus Loerke Justus Loerke UltraStrukturNetzwerk, Max Planck Institute for Molecular Genetics, Berlin, Germany Search for more papers by this author Thorsten Mielke Thorsten Mielke UltraStrukturNetzwerk, Max Planck Institute for Molecular Genetics, Berlin, Germany Search for more papers by this author Wei Zhang Wei Zhang Department of Biochemistry and Molecular Biology, The University of Texas—Houston Medical School, Houston, TX, USA Search for more papers by this author Pawel A Penczek Pawel A Penczek Department of Biochemistry and Molecular Biology, The University of Texas—Houston Medical School, Houston, TX, USA Search for more papers by this author V Ramakrishnan Corresponding Author V Ramakrishnan Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Christian M T Spahn Corresponding Author Christian M T Spahn Institut für Medizinische Physik und Biophysik, Charite-Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Jan-Christian Schuette Jan-Christian Schuette Institut für Medizinische Physik und Biophysik, Charite-Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Frank V Murphy IV Frank V Murphy IV Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Ann C Kelley Ann C Kelley Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author John R Weir John R Weir Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Jan Giesebrecht Jan Giesebrecht Institut für Medizinische Physik und Biophysik, Charite-Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Sean R Connell Sean R Connell Institut für Medizinische Physik und Biophysik, Charite-Universitätsmedizin Berlin, Berlin, GermanyPresent address: Institut für Organische Chemie, JW Goethe Universität, Max-von-Laue Strasse 7, 60438 Frankfurt am Main, Germany Search for more papers by this author Justus Loerke Justus Loerke UltraStrukturNetzwerk, Max Planck Institute for Molecular Genetics, Berlin, Germany Search for more papers by this author Thorsten Mielke Thorsten Mielke UltraStrukturNetzwerk, Max Planck Institute for Molecular Genetics, Berlin, Germany Search for more papers by this author Wei Zhang Wei Zhang Department of Biochemistry and Molecular Biology, The University of Texas—Houston Medical School, Houston, TX, USA Search for more papers by this author Pawel A Penczek Pawel A Penczek Department of Biochemistry and Molecular Biology, The University of Texas—Houston Medical School, Houston, TX, USA Search for more papers by this author V Ramakrishnan Corresponding Author V Ramakrishnan Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Christian M T Spahn Corresponding Author Christian M T Spahn Institut für Medizinische Physik und Biophysik, Charite-Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Author Information Jan-Christian Schuette1,‡, Frank V Murphy2,‡, Ann C Kelley2, John R Weir2, Jan Giesebrecht1, Sean R Connell1, Justus Loerke3, Thorsten Mielke3, Wei Zhang4, Pawel A Penczek4, V Ramakrishnan 2 and Christian M T Spahn 1 1Institut für Medizinische Physik und Biophysik, Charite-Universitätsmedizin Berlin, Berlin, Germany 2Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, UK 3UltraStrukturNetzwerk, Max Planck Institute for Molecular Genetics, Berlin, Germany 4Department of Biochemistry and Molecular Biology, The University of Texas—Houston Medical School, Houston, TX, USA ‡These authors contributed equally to this work *Corresponding authors: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK. Tel.: +44 1223 402213; Fax: +44 1223 213556; E-mail: [email protected] of Medical Physics and Biophysics, Charite-Universitätsmedizin Berlin, Ziegelstrasse 5-9, 10117 Berlin, Germany. Tel.: +49 30 450 5241 31; Fax: +49 30 450 5249 31; E-mail: [email protected] The EMBO Journal (2009)28:755-765https://doi.org/10.1038/emboj.2009.26 There is a Have you seen ...? (March 2009) associated with this Article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have used single-particle reconstruction in cryo-electron microscopy to determine a structure of the Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF-Tu), tRNA and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin. This represents the state in the decoding process just after codon recognition by tRNA and the resulting GTP hydrolysis by EF-Tu, but before the release of EF-Tu from the ribosome. Progress in sample purification and image processing made it possible to reach a resolution of 6.4 Å. Secondary structure elements in tRNA, EF-Tu and the ribosome, and even GDP and kirromycin, could all be visualized directly. The structure reveals a complex conformational rearrangement of the tRNA in the A/T state and the interactions with the functionally important switch regions of EF-Tu crucial to GTP hydrolysis. Thus, the structure provides insights into the molecular mechanism of signalling codon recognition from the decoding centre of the 30S subunit to the GTPase centre of EF-Tu. Introduction Ribosomes are large macromolecular machines that translate the genetic message to make proteins. Although the ribosome itself is at the core of this process, several protein factors have important functions in each of the four broad stages of translation, that is, initiation, elongation, termination and ribosome recycling (Ramakrishnan, 2002; Frank and Spahn, 2006). Many of these factors are GTPases, among them initiation factor IF2, elongation factors EF-Tu (ternary complex of elongation factor Tu), EF-G and release factor RF3. Thus, GTP hydrolysis by protein factors is an essential part of translation in each of the stages of translation. Among the GTPase translation factors, EF-Tu has an essential function in tRNA selection during decoding. EF-Tu in the GTP-bound form has high affinity for aminoacyl tRNAs (aa-tRNAs). The ternary complex of EF-Tu, aa-tRNA and GTP binds to the ribosomal A-site in the initial step of decoding. A cognate interaction between the codon on mRNA and anticodon on tRNA in the A-site leads to GTP hydrolysis by EF-Tu and subsequent dissociation of EF-Tu•GDP from the ribosome. The structures of isolated EF-Tu in complex with GDP or the GTP analogue GDPNP, as well as its ternary complex with GTP and aa-tRNA, have all been characterized in molecular detail (for review, see Hilgenfeld, 1995; Andersen et al, 2003). The hydrolysis of GTP and the release of the gamma phosphate induce a major rearrangement of the universally conserved switch I (effector loop) and switch II regions, which in turn results in a gross conformational change in EF-Tu. The global change also results in a strong reduction in affinity for aa-tRNA of the GDP-bound form. How the ribosome induces GTP hydrolysis by EF-Tu is a major question in translation. Kinetic, structural, genetic and biochemical data over the last decade have established that decoding is a complicated multistep process in which an active role is played by the ribosome itself (Rodnina and Wintermeyer, 2001; Ogle and Ramakrishnan, 2005; Frank and Spahn, 2006; Korostelev et al, 2008; Marshall et al, 2008). Kinetic work shows that a rapid, reversible, initial binding of the ternary complex is followed by a slower codon recognition step (Rodnina and Wintermeyer, 2001; Marshall et al, 2008). Codon recognition by cognate (but not near-cognate) tRNA leads to an acceleration of GTPase activation and GTP hydrolysis. This suggests that binding of cognate tRNA induces conformational changes in the ribosomal complex required for GTP hydrolysis by EF-Tu. GTP hydrolysis is followed by EF-Tu release and movement (accommodation) of the tRNA into the peptidyl transferase centre, after which peptide bond formation occurs rapidly. The antibiotic kirromycin traps the process after GTP hydrolysis but before EF-Tu release. A number of biochemical and structural studies can be correlated directly with the kinetic data. During codon recognition, the correctness of the codon–anticodon interaction is checked by interactions of three conserved bases of 16S RNA at the decoding centre of the 30S subunit with the minor groove of the codon–anticodon base pairs (Ogle et al, 2001). Successful codon–anticodon recognition by the 30S subunit leads to a conformational change in the 30S subunit that would move the shoulder of the subunit closer to EF-Tu (Ogle et al, 2002). This led to the view that the additional energy obtained from the minor groove interactions of the ribosome with cognate tRNA at the decoding centre led to a domain closure of the ribosome essential for GTP hydrolysis by EF-Tu and subsequent steps of successful decoding (Ogle et al, 2002; Ogle and Ramakrishnan, 2005). Early chemical footprinting studies suggested the existence of an A/T state as long as EF-Tu was present on the ribosome, in which the aminoacyl end of the incoming tRNA cannot enter the peptidyl transferase centre until GTP hydrolysis occurs and EF-Tu has been released, thus ensuring that decoding has taken place prior to peptide bond formation (Moazed and Noller, 1989). Single-particle cryo-EM studies of an EF-Tu ribosome complex stalled with kirromycin (similar to the one reported here) have directly revealed the existence of the A/T state (Stark et al, 1997). In this A/T state, the aminoacyl end of the tRNA is still in contact with its binding pocket in EF-Tu and thus unable to enter the peptidyl transferase centre, but the anticodon of the tRNA is in the decoding centre of the 30S subunit. Later higher resolution studies of the same complex (Stark et al, 2002; Valle et al, 2002, 2003) showed that the A/T state was characterized by a distorted tRNA as well as changes in the conformation of EF-Tu and the ribosome. Indeed, distortions of the tRNA during decoding have long been suggested as important in decoding (Yarus and Smith, 1995). Although cryo-EM has provided important structural information about the kirromycin-stalled decoding complex, previous cryo-EM maps have been obtained at low to intermediate resolution (20–10 Å). At this resolution, detailed interpretation of maps in molecular terms is problematic; indeed, there are several discrepancies in the interpretation of previous cryo-EM maps of this complex (Stark et al, 1997, 2002; Valle et al, 2002, 2003). At about 7 Å, it becomes possible to directly visualize double helices of RNA as well as alpha helices of proteins, thus making placement of individual domains far more accurate and allowing a more detailed analysis of molecular interactions and conformational changes, as demonstrated by Petry et al (2005) in their interpretation of X-ray crystallographic maps of the ribosome. However, X-ray crystallography of functional ribosomal complexes is still very challenging. Despite considerable progress in the structural analysis of several ribosomal complexes at atomic or near-atomic resolution, no crystal structure of a GTPase factor bound to the ribosome has yet been reported. We present here a cryo-EM map of the Thermus thermophilus 70S ribosome bound to the ternary complex (EF-Tu•Phe-tRNA•GTP, stalled with kirromycin) at a resolution of 6.4 Å at 0.5 cutoff of the Fourier shell correlation (FSC) criterion, with significant information extending to 4.7 Å, as determined by the 3σ criterion (Orlova et al, 1997). At this resolution, we can directly visualize not only secondary structure elements such as α-helices as well as the RNA backbone but also even ligands such as GDP and kirromycin. Moreover, the crystal structure of the T. thermophilus ribosome at 2.8-Å resolution (Selmer et al, 2006) greatly facilitated interpretation of the cryo-EM map in molecular terms, which revealed important molecular interactions and conformational changes. The structure reveals a complex rearrangement of the tRNA and provides a detailed view of the interaction of the GTPase centre of EF-Tu and especially the switch regions with the ribosome. The structure provides insights into how successful recognition of the cognate codon is signalled from the decoding centre of the 30S subunit to the GTPase centre of EF-Tu about 80 Å away, and leads to a model of how the ribosome might stimulate the GTPase activity of translational GTPases. Results and discussion Intrinsic conformational heterogeneity of the ribosomal decoding complex and progression to higher resolution A general problem with ribosomal complexes is achieving equal stoichiometry of all the components. To avoid this problem and generate the most homogeneous complex possible, we used an affinity tag method previously described for the crystallography of release factor complexes (Petry et al, 2005). In this procedure, ternary complex containing His-tagged EF-Tu was bound to programmed ribosomes containing tRNAfMet in the P-site in the presence of kirromycin. The entire ribosomal complex was affinity purified on a nickel column using the His-tag on EF-Tu. As previously demonstrated for release factors, only the ribosome complex, which had EF-Tu and tRNA, that was cognate for the A-site codon was retained on the nickel column. However, although the specimen behaved quite well during initial phases of image reconstruction and always exhibited strong density for the ternary complex, the maps calculated from the full data set showed indications of conformational heterogeneity as we progressed to sub-nanometer resolution. For example, the L7/L12 stalk base region of the ribosome was strongly disordered and had a fragmented appearance. To obtain a more homogeneous population of particle images, we classified the data set (586 329 particle images) using an unsupervised '3D K-means procedure' (Penczek et al, 2006) and obtained a major sub-population (I) consisting of 55% of the particles (Supplementary Figure S1). Interestingly, cryo-EM maps calculated from all of the minor sub-populations of particle images exhibit density for the ternary complex (Supplementary Figures S1 and S2). The behaviour of the data set is thus different from those of unpurified ribosomal complexes, where a classification procedure resulted in a significant fraction of ribosomes lacking the ligand (for example, Valle et al, 2002; Penczek et al, 2006; Connell et al, 2007) and directly corroborates the success of our affinity purification using the His-tagged EF-Tu. The 70S•tRNA•EF-Tu•GDP•kirromycin complex at 6.4-Å resolution The major sub-population, consisting of 323 688 particles, was used to obtain an improved cryo-EM map (Figure 1A; Supplementary Movie 1) at the unprecedented resolution of 6.4 Å by the conservative 0.5 cutoff of the FSC criterion (Supplementary Figure S3). As would be expected from the increased resolution, the map reveals significantly more details than previous maps of the ribosomal decoding complex at low to intermediate resolution (Stark et al, 1997, 2002; Valle et al, 2002, 2003) or even previous cryo-EM maps of ribosomal complexes at 7 to 8-Å resolution (Schüler et al, 2006; Connell et al, 2007). Not only are α-helical secondary structure elements of proteins clearly resolved but also density for unstructured peptide chains corresponding to the extended tails of ribosomal proteins (Figure 2; Supplementary Figure S4). Distinct density is present even for the low-molecular ligands GDP (Figure 2B) and the antibiotic kirromycin (Figure 2C). However, β-sheets are not fully resolved: they appear as flat surfaces and only occasionally does the density indicate a separation of strands. This improved cryo-EM map enables a more detailed analysis of the interactions of EF-Tu and tRNA with the ribosome. Figure 1.Overview of the 70S•EF-Tu•Phe-tRNA•GDP•kirromycin complex. A surface representation of the cryo-EM map is shown (A) from the top; (B) from the L7/L12 side; (C) from the 30S side, with 30S removed and (D) from the 50S side, with 50S removed. The components are coloured distinctly (30S subunit, yellow; 50S subunit, blue; EF-Tu, red; A/T-tRNA, orange; P-tRNA, green; E-tRNA, brown; mRNA, pink). Download figure Download PowerPoint Figure 2.Details seen in the electron density map at 5.7- to 6.4-Å resolution. (A) Overall structure of the ternary complex showing the interactions between the EF-Tu and A/T tRNA. The density for the ternary complex has been computationally separated using a mask generous enough to show the sites of interaction with the ribosome. At this resolution, secondary structure elements of RNA and protein are clearly distinguishable. (B) A region of EF-Tu showing additional density that corresponds to GDP. (C) Density for the low-molecular weight kirromycin seen between the domains of EF-Tu. Download figure Download PowerPoint Overall, the conformation of the 70S•tRNA•EF-Tu•GDP•kirromycin complex is similar to the crystal structure of the 70S ribosome complex with mRNA and tRNAs. A good fit could be obtained by docking the atomic model of the T. thermophilus ribosome (Selmer et al, 2006) as three rigid bodies corresponding to the main body of the 50S subunit and the head and the body/platform domains of the 30S subunit. In addition, the L1 and L7/L12 stalks required further adjustment. Apart from the ribosome itself, atomic models of mRNA and the P- and E-site tRNAs derived from the crystal structure of the 70S ribosome from T. thermophilus in complex with mRNA and tRNAs (Selmer et al, 2006) could also readily be docked into the cryo-EM map. A molecular model for the ribosome-bound ternary complex was derived from the X-ray structures of the ribosome-bound anticodon stem loop of the A-site tRNA (Yusupov et al, 2001; Selmer et al, 2006), the ternary complex (Nissen et al, 1995) and EF-Tu•GDP in complex with methylkirromycin (also called aurodox; Vogeley et al, 2001) as detailed below. Thus, we could derive a nearly complete molecular model for the kirromycin-stalled ribosomal decoding complex. A complex rearrangement in the A/T tRNA It is clear that the tRNA in the A/T state has to be deformed in comparison to the accommodated A-site tRNA to allow codon–anticodon interaction in the decoding centre to occur while the acceptor stem and the aminoacyl-end are still bound to EF-Tu. A distortion of the anticodon loop was proposed to account for the difference in orientation of accommodated A tRNA and A/T tRNA in one cryo-EM study (Stark et al, 2002). In other cryo-EM studies, a kink-like deformation of the A/T tRNA was proposed that occurs at the junction between the anticodon and D stems (Valle et al, 2002, 2003). No deformation of the anticodon loop can be observed in our present cryo-EM map, and the X-ray structures of the anticodon stem loops together with the A-site mRNA codon (Selmer et al, 2006) can be readily fit into the cryo-EM density map (Figure 2A). A kink-like deformation of the A/T tRNA at the junction between the anticodon and D stems can be clearly seen in our map in good overall agreement with earlier observations (Valle et al, 2003). However, the exact nature of the tRNA deformation could not be determined precisely at the resolution of the previous cryo-EM studies, and the deformation was modelled as smoothly distributed over the four base pairs surrounding the junction (Valle et al, 2003). In contrast, in our present map, we do not observe a significant change of the upper base pairs of the anticodon stem or the lower base pairs of the D stem. Moreover, it was not possible to fit the density by docking the T-acceptor arm together with the D stem and D loop of the tRNA as a single rigid body. Independent rigid-body docking of the T-acceptor arm and the D stem (together with parts of the D loop and the variable loop) leads to an improved fit for the A/T tRNA (Figures 2 and 3). As a result, the D loop is rotated relative to the T-acceptor arm such that the angle between both arms of the L-shaped tRNA is opened in the A/T tRNA compared with the canonical tRNA structure. This rotation of the D stem is expected to have an effect on the conformation of the D loop, which stabilizes the relative positioning of the T stem loop and D stem by a tertiary interaction with the T loop. Indeed, the present cryo-EM density accounts for G18 and G19, which directly interact with the T loop, but the linker regions including positions 16, 17 and 20 appear to be disordered. This observed change in the D loop conformation explains a long-standing observation from kinetic data on decoding (Rodnina et al, 1994), which used a tRNA with proflavin at positions 16 and 17 as a reporter. The fluorescence from this reporter showed a marked temporal increase during decoding, consistent with the observed disorder in the D loop. Figure 3.Distortions in the tRNA during decoding. (A) A molecular model for the A/T-tRNA derived by fitting various segments of the tRNA separately into the electron density. The region of disorder in the D loop is highlighted (green circle, missing nucleotides are labelled) and is consistent with fluorescence changes in a reporter in this loop (see text for details). (B, C) Superposition of the distorted A/T-tRNA (coloured ribbon: acceptor stem, orange; T stem and T loop, cyan; D stem with variable loop, green; D loop, yellow; anticodon stem loop, magenta) and canonical tRNA (grey ribbon). The superpositions were carried out using the acceptor arm (B) and the anticodon stem loop (C), and show that in addition to the kink between the D stem and anticodon stem loop, there is also a rotation of the D loop and stem relative to the acceptor arm. Download figure Download PowerPoint The opening of the tRNA at the junction between the D stem and T-acceptor arm moves the anticodon loop away from the position of the mRNA codon. To facilitate codon–anticodon interactions by counteracting this movement, the kink between the D and anticodon stems has to be even larger than previously proposed (Valle et al, 2003). Indeed, if we superpose atomic models for the tRNAs at the D stem, the distance between G34 of the anticodon loop between the A/A tRNA (Yusupov et al, 2001) and the previous model of the A/T tRNA (Valle et al, 2003) is 12.5 Å, and this is increased to 18.0 Å for the present structure of the A/T tRNA. The exact conformation of the A/T tRNA at the kink cannot be described in atomic terms even at the improved resolution of the current cryo-EM map, but it is in good agreement with local conformational changes of the unpaired nucleotides between the anticodon and D stems, for example, G26 and G45, which was suggested as a conformational hinge point when the structure of tRNA was originally determined (Robertus et al, 1974). The nearby Hirsh suppressor mutation at position 24 in the D stem (Hirsh, 1971) was recently shown to increase the rate of GTP hydrolysis in decoding (Cochella and Green, 2005). Presumably mutating this residue alters the properties of the neighbouring residues, facilitating a hinge movement in tRNA, thus allowing the transition state to be reached even for incorrect tRNAs. The angle of twist between the acceptor stem and T stem of the tRNA is greater in the ternary complex with a GTP analogue than it is in free tRNA (Nissen et al, 1995). In the presence of antibiotics enacyloxin IIa or kirromycin, this twist is partially relieved and the conformation of the tRNA within the ternary complex is more similar to the conformation of free tRNA (Parmeggiani et al, 2006). The untwisted T-acceptor arm (without the 3′-CCA end, which was fit together with EF-Tu) from the ribosome-bound A/A tRNA (Yusupov et al, 2001) fits slightly better into the cryo-EM map than the one from the kirromycin-containing ternary complex (PDB identifier 1OB2; cross-correlation coefficient of 0.80 versus 0.78) and we used it for our final model of the A/T tRNA (Figures 2, 3 and 4). This difference is small and may not be significant at the present resolution of our cryo-EM map, but the larger twist present in the ternary complex in the absence of antibiotics (Nissen et al, 1995) is relieved during the events of ribosomal decoding and a conformational change of the T-acceptor arm of tRNA might be part of the mechanism leading to EF-Tu dissociation from the tRNA. Figure 4.Structures of the ternary complex on and off the ribosome. Superposition of the molecular model for the ribosome-bound ternary complex (red ribbons) with (A) the X-ray structure of the kirromycin-containing ternary complex (PDB identifier 1OB2, green ribbons) or (B) the X-ray structure of the ternary complex (Nissen et al, 1995) (turquoise ribbons). The ternary complexes are aligned at domain I (G domain) of EF-Tu. Download figure Download PowerPoint In summary, if we consider the X-ray structure of the ternary complex to represent the structure of unbound ternary complex in solution (Nissen et al, 1995) and if we assume that the present model of the kirromycin-stalled ribosome-bound ternary complex represents a state that follows but is close to the transition state for GTP hydrolysis during decoding, the tRNA undergoes conformational rearrangements that are far more complex than previously anticipated (Figures 3 and 4): (i) the twist between the T and acceptor stems is relieved, (ii) the region between the T-acceptor arm and D stem is opened, (iii) accompanied by a partial unfolding of the D loop and (iv) a kink is introduced at the junction between the D and anticodon stems. These rearrangements that occur throughout the whole tRNA molecule are consistent with a direct role of tRNA in signalling successful codon recognition to the GTPase centre on EF-Tu (Piepenburg et al, 2000). The structure of ribosome-bound EF-Tu•GDP•kirromycin Not surprisingly, EF-Tu on the ribosome has a distinct conformation that is different from the GTP- or GDP-bound forms of the isolated structure. The EF-Tu•GDP•methylkirromycin structure (Vogeley et al, 2001) could be docked readily into the cryo-EM density as a single rigid body (Figure 2) in contrast to the EF-Tu•GMPPNP structure (Berchtold et al, 1993) (Supplementary data). Methylkirromycin is functionally equivalent to kirromycin and prevents the large conformational change of EF-Tu upon GTP hydrolysis by gluing together domains I and III. However, the two structures are not equivalent: in the methylkirromycin structure, domains II and III are rotated relative to domain I by about 10° and 15°, respectively (Vogeley et al, 2001). The docking shows that the conformation of EF-Tu in complex with GDP and methylkirromycin (Vogeley et al, 2001) is very similar to the structure of EF-Tu in the kirromycin-stalled, ribosome-bound ternary complex. This result is at variance with the cryo-EM analysis of Stark et al (2002) on this complex, who could not obtain a good fit with the EF-Tu•GDP•methylkirromycin structure and instead proposed a large movement of domain III of EF-Tu. In contrast, Escherichia coli EF-Tu from the X-ray structure of the kirromycin-containing Phe-tRNAPhe•EF-Tu•GMPPNP•kirromycin complex (PDB code: 1OB2) could be docked as a single rigid body into the previous cryo-EM map of Valle et al (2003) and this is in agreement with our findings presented here, because the latter EF-Tu structure is very similar to the EF-Tu•GDP•methylkirromycin structure. In agreement with lower resolution cryo-EM studies (Stark et al, 1997; Valle et al, 2003), the relative orientation of tRNA and EF-Tu is changed in the ribosome-bound ternary complex relative to the X-ray structures of ternary complex (Nissen et al, 1995) or the Phe-tRNAPhe•EF-Tu•GMPPNP•kirromycin complex (PDB code: 1OB2). The changes can be described as a small rotation of EF-Tu and a translational movement in the direction from the acceptor stem towards the T loop of the tRNA (Figure 4). This movement of EF-Tu is partially counteracted by the release of the twist between the acceptor and T stems of the tRNA that is present in the unbound ternary complex (Nissen et al, 1995). At the T stem, however, domain III of EF-Tu moves 5–6 Å relative to the tRNA while maintaining extensive interactions with it (
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