Abstract
In protein synthesis, elongation factor G (EF-G) facilitates movement of tRNA–mRNA by one codon, which is coupled to the ratchet-like rotation of the ribosome complex and is triggered by EF-G–mediated GTP hydrolysis. Here we report the structure of a pretranslocational ribosome bound to Thermus thermophilus EF-G trapped with a GTP analog. The positioning of the catalytic His87 into the active site coupled to hydrophobic-gate opening involves the 23S rRNA sarcin-ricin loop and domain III of EF-G and provides a structural basis for the GTPase activation of EF-G. Interactions of the hybrid peptidyl-site–exit-site tRNA with ribosomal elements, including the entire L1 stalk and proteins S13 and S19, shed light on how formation and stabilization of the hybrid tRNA is coupled to head swiveling and body rotation of the 30S as well as to closure of the L1 stalk.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Change history
12 January 2015
In view of the differences between our findings reported in this study and in Tourigny et al. (Science 340, 1235490, 2013), we have rerefined the structure with Refmac (Murshudov, G.N. et al., Acta Crystallogr. D Biol. Crystallogr. 67, 355–367, 2011). This resulted in better statistics and revealed that the structure contained tRNAPhe in the P/E site, as in Tourigny et al., and not tRNAfMet as we had originally interpreted. (Both tRNAfMet and tRNAPhe had been included for crystallization.) Thus, the structure reported here is essentially identical to that reported in Tourigny et al., and we thank D. S. Tourigny, G. Murshudov and V. Ramakrishnan for alerting us about this issue after they had rerefined our data with Refmac. We have updated the coordinates accordingly (PDB 4CR1) and made the following corrections: (i) on pages 1078 and page 1080, tRNAfMet was changed to tRNAPhe; (ii) on page 1081, including Figure 4b, A43 was changed to C43, and A43-U27 was changed to C43-G27; (iii) on page 1082, G42 was changed to C42. The text and Figure 4 have been updated to reflect the revised information as of 12 January 2015.
References
Moazed, D. & Noller, H.F. Intermediate states in the movement of transfer RNA in the ribosome. Nature 342, 142–148 (1989).
Frank, J. & Agrawal, R.K. A ratchet-like inter-subunit reorganization of the ribosome during translocation. Nature 406, 318–322 (2000).
Rodnina, M.V., Savelsbergh, A., Katunin, V.I. & Wintermeyer, W. Hydrolysis of GTP by elongation factor G drives tRNA movement on the ribosome. Nature 385, 37–41 (1997).
Connell, S.R. et al. Structural basis for interaction of the ribosome with the switch regions of GTP-bound elongation factors. Mol. Cell 25, 751–764 (2007).
Schuette, J.C. et al. GTPase activation of elongation factor EF-Tu by the ribosome during decoding. EMBO J. 28, 755–765 (2009).
Villa, E. et al. Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis. Proc. Natl. Acad. Sci. USA 106, 1063–1068 (2009).
Voorhees, R.M., Schmeing, T.M., Kelley, A.C. & Ramakrishnan, V. The mechanism for activation of GTP hydrolysis on the ribosome. Science 330, 835–838 (2010).
Liljas, A., Ehrenberg, M. & Aqvist, J. Comment on “The mechanism for activation of GTP hydrolysis on the ribosome”. Science 333, 37 (2011).
Jin, H., Kelley, A.C. & Ramakrishnan, V. Crystal structure of the hybrid state of ribosome in complex with the guanosine triphosphatase release factor 3. Proc. Natl. Acad. Sci. USA 108, 15798–15803 (2011).
Zhou, J., Lancaster, L., Trakhanov, S. & Noller, H.F. Crystal structure of release factor RF3 trapped in the GTP state on a rotated conformation of the ribosome. RNA 18, 230–240 (2012).
Valle, M. et al. Locking and unlocking of ribosomal motions. Cell 114, 123–134 (2003).
Taylor, D.J. et al. Structures of modified eEF2 80S ribosome complexes reveal the role of GTP hydrolysis in translocation. EMBO J. 26, 2421–2431 (2007).
Selmer, M., Gao, Y.G., Weixlbaumer, A. & Ramakrishnan, V. Ribosome engineering to promote new crystal forms. Acta Crystallogr. D Biol. Crystallogr. 68, 578–583 (2012).
Gao, Y.G. et al. The structure of the ribosome with elongation factor G trapped in the posttranslocational state. Science 326, 694–699 (2009).
Trabuco, L.G. et al. The role of L1 stalk-tRNA interaction in the ribosome elongation cycle. J. Mol. Biol. 402, 741–760 (2010).
Lill, R., Robertson, J.M. & Wintermeyer, W. Binding of the 3′ terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation. EMBO J. 8, 3933–3938 (1989).
Fei, J. et al. Allosteric collaboration between elongation factor G and the ribosomal L1 stalk directs tRNA movements during translation. Proc. Natl. Acad. Sci. USA 106, 15702–15707 (2009).
Dorner, S., Brunelle, J.L., Sharma, D. & Green, R. The hybrid state of tRNA binding is an authentic translation elongation intermediate. Nat. Struct. Mol. Biol. 13, 234–241 (2006).
Ratje, A.H. et al. Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites. Nature 468, 713–716 (2010).
Dunkle, J.A. et al. Structures of the bacterial ribosome in classical and hybrid states of tRNA binding. Science 332, 981–984 (2011).
Ben-Shem, A. et al. The structure of the eukaryotic ribosome at 3.0 A resolution. Science 334, 1524–1529 (2011).
Jenner, L., Demeshkina, N., Yusupova, G. & Yusupov, M. Structural rearrangements of the ribosome at the tRNA proofreading step. Nat. Struct. Mol. Biol. 17, 1072–1078 (2010).
Czworkowski, J., Wang, J., Steitz, T.A. & Moore, P.B. The crystal structure of elongation factor G complexed with GDP, at 2.7 A resolution. EMBO J. 13, 3661–3668 (1994).
Hansson, S., Singh, R., Gudkov, A.T., Liljas, A. & Logan, D.T. Structural insights into fusidic acid resistance and sensitivity in EF-G. J. Mol. Biol. 348, 939–949 (2005).
Hausner, T.P., Atmadja, J. & Nierhaus, K.H. Evidence that the G2661 region of 23S rRNA is located at the ribosomal binding sites of both elongation factors. Biochimie 69, 911–923 (1987).
Rambelli, F. et al. Effect of the antibiotic purpuromycin on cell-free protein-synthesizing systems. Biochem. J. 259, 307–310 (1989).
Clementi, N., Chirkova, A., Puffer, B., Micura, R. & Polacek, N. Atomic mutagenesis reveals A2660 of 23S ribosomal RNA as key to EF-G GTPase activation. Nat. Chem. Biol. 6, 344–351 (2010).
AEvarsson, A. et al. Three-dimensional structure of the ribosomal translocase: elongation factor G from Thermus thermophilus. EMBO J. 13, 3669–3677 (1994).
Martemyanov, K.A. & Gudkov, A.T. Domain III of elongation factor G from Thermus thermophilus is essential for induction of GTP hydrolysis on the ribosome. J. Biol. Chem. 275, 35820–35824 (2000).
Savelsbergh, A. et al. An elongation factor G-induced ribosome rearrangement precedes tRNA-mRNA translocation. Mol. Cell 11, 1517–1523 (2003).
Savelsbergh, A., Mohr, D., Kothe, U., Wintermeyer, W. & Rodnina, M.V. Control of phosphate release from elongation factor G by ribosomal protein L7/12. EMBO J. 24, 4316–4323 (2005).
Feng, S., Chen, Y. & Gao, Y.G. Crystal structure of 70S ribosome with both cognate tRNAs in the E and P sites representing an authentic elongation complex. PLoS ONE 8, e58829 (2013).
Munro, J.B., Altman, R.B., O'Connor, N. & Blanchard, S.C. Identification of two distinct hybrid state intermediates on the ribosome. Mol. Cell 25, 505–517 (2007).
Pan, D., Kirillov, S., Zhang, C.M., Hou, Y.M. & Cooperman, B.S. Rapid ribosomal translocation depends on the conserved 18–55 base pair in P-site transfer RNA. Nat. Struct. Mol. Biol. 13, 354–359 (2006).
Sprinzl, M. et al. Regulation of GTPases in the bacterial translation machinery. Biol. Chem. 381, 367–375 (2000).
Wallin, G., Kamerlin, S.C. & Aqvist, J. Energetics of activation of GTP hydrolysis on the ribosome. Nat. Commun. 4, 1733 (2013).
Schweins, T. et al. Substrate-assisted catalysis as a mechanism for GTP hydrolysis of p21ras and other GTP-binding proteins. Nat. Struct. Biol. 2, 36–44 (1995).
Cukras, A.R., Southworth, D.R., Brunelle, J.L., Culver, G.M. & Green, R. Ribosomal proteins S12 and S13 function as control elements for translocation of the mRNA:tRNA complex. Mol. Cell 12, 321–328 (2003).
Cukras, A.R. & Green, R. Multiple effects of S13 in modulating the strength of intersubunit interactions in the ribosome during translation. J. Mol. Biol. 349, 47–59 (2005).
Joseph, S. & Noller, H.F. EF-G-catalyzed translocation of anticodon stem-loop analogs of transfer RNA in the ribosome. EMBO J. 17, 3478–3483 (1998).
Agirrezabala, X. et al. Visualization of the hybrid state of tRNA binding promoted by spontaneous ratcheting of the ribosome. Mol. Cell 32, 190–197 (2008).
Sengupta, J. et al. Visualization of the eEF2–80S ribosome transition-state complex by cryo-electron microscopy. J. Mol. Biol. 382, 179–187 (2008).
Tourigny, D.S., Fernandez, I.S., Kelley, A.C. & Ramakrishnan, V. Elongation factor G bound to the ribosome in an intermediate state of translocation. Science 340, 1235490 (2013).
Pulk, A. & Cate, J.H. Control of ribosomal subunit rotation by elongation factor G. Science 340, 1235970 (2013).
Zhou, J., Lancaster, L., Donohue, J.P. & Noller, H.F. Crystal structures of EF-G-ribosome complexes trapped in intermediate states of translocation. Science 340, 1236086 (2013).
Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–200 (1993).
McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Brünger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
Collaborative Computational Project Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).
Acknowledgements
The mutant T. thermophilus strain was provided by V. Ramakrishnan's group, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.We thank C. Neubauer for comments on the manuscript and M. Wang for help with data collection. This work was supported by Singapore National Research Foundation NRF-RF2009-RF001-267 (Y.-G.G.) and a Nanyang Technological University Startup grant (Y.-G.G.).
Author information
Authors and Affiliations
Contributions
Y.-G.G. designed the study; Y.C., S.F. and Y.-G.G. performed the preliminary experiment; Y.C. and Y.-G.G. optimized crystallization and carried out data collection; Y.-G.G., Y.C, V.K. and R.E. performed model building, refinement and data analysis; Y.-G.G., Y.C. and R.E. wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–5 and Supplementary Tables 1 and 2 (PDF 1299 kb)
Rights and permissions
About this article
Cite this article
Chen, Y., Feng, S., Kumar, V. et al. Structure of EF-G–ribosome complex in a pretranslocation state. Nat Struct Mol Biol 20, 1077–1084 (2013). https://doi.org/10.1038/nsmb.2645
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.2645
This article is cited by
-
Distinct mechanisms of the human mitoribosome recycling and antibiotic resistance
Nature Communications (2021)
-
Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features of mitochondrial translation elongation
Nature Communications (2020)
-
Recurring RNA structural motifs underlie the mechanics of L1 stalk movement
Nature Communications (2017)
-
Effect of Fusidic Acid on the Kinetics of Molecular Motions During EF-G-Induced Translocation on the Ribosome
Scientific Reports (2017)
-
A target-protection mechanism of antibiotic resistance at atomic resolution: insights into FusB-type fusidic acid resistance
Scientific Reports (2016)