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mRNA translocation occurs during the second step of ribosomal intersubunit rotation

Nature Structural & Molecular Biology volume 18, pages 457–462 (2011)Cite this article

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Abstract

During protein synthesis, mRNA and tRNA undergo coupled translocation through the ribosome in a process that is catalyzed by elongation factor G (EF-G). On the basis of cryo-EM reconstructions, counterclockwise and clockwise rotational movements between the large and small ribosomal subunits have been implicated in a proposed ratcheting mechanism to drive the unidirectional movement of translocation. We used a combination of two fluorescence-based approaches to study the timing of these events, intersubunit fluorescence resonance energy transfer measurements to observe relative rotational movement of the subunits, and a fluorescence quenching assay to monitor translocation of mRNA. Binding of EF-G–GTP first induces rapid counterclockwise intersubunit rotation, followed by a slower, clockwise reversal of the rotational movement. We compared the rates of these movements and found that mRNA translocation occurs during the second, clockwise rotation event, corresponding to the transition from the hybrid state to the classical state.

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Figure 1: Hybrid-state model of translocation and experimental design.
Figure 2: Kinetics of intersubunit rotation and mRNA translocation.
Figure 3: Resolution of the first and second steps of intersubunit rotation with antibiotics.
Figure 4: Stabilization of the rotated, hybrid-state conformation by binding of EF-G–GDP in the presence of fusidic acid.
Figure 5: Effect of inhibition of EF-G release on the second (clockwise) step of intersubunit rotation and mRNA translocation.

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References

  1. Cate, J.H., Yusupov, M.M., Yusupova, G.Z., Earnest, T.N. & Noller, H.F. X-ray crystal structures of 70S ribosome functional complexes. Science 285, 2095–2104 (1999).

    Article  CAS  PubMed  Google Scholar 

  2. Korostelev, A., Trakhanov, S., Laurberg, M. & Noller, H.F. Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements. Cell 126, 1065–1077 (2006).

    Article  CAS  PubMed  Google Scholar 

  3. Selmer, M. et al. Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313, 1935–1942 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Yusupov, M.M. et al. Crystal structure of the ribosome at 5.5 Å resolution. Science 292, 883–896 (2001).

    Article  CAS  PubMed  Google Scholar 

  5. Yusupova, G.Z., Yusupov, M.M., Cate, J.H. & Noller, H.F. The path of messenger RNA through the ribosome. Cell 106, 233–241 (2001).

    Article  CAS  PubMed  Google Scholar 

  6. Moazed, D. & Noller, H.F. Intermediate states in the movement of transfer RNA in the ribosome. Nature 342, 142–148 (1989).

    Article  CAS  PubMed  Google Scholar 

  7. Spiegel, P.C., Ermolenko, D.N. & Noller, H.F. Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome. RNA 13, 1473–1482 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Pan, D., Kirillov, S.V. & Cooperman, B.S. Kinetically competent intermediates in the translocation step of protein synthesis. Mol. Cell 25, 519–529 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Frank, J. & Agrawal, R.K. A ratchet-like inter-subunit reorganization of the ribosome during translocation. Nature 406, 318–322 (2000).

    Article  CAS  PubMed  Google Scholar 

  10. Gao, H. et al. Study of the structural dynamics of the E. coli 70S ribosome using real-space refinement. Cell 113, 789–801 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Valle, M. et al. Locking and unlocking of ribosomal motions. Cell 114, 123–134 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Frank, J., Gao, H., Sengupta, J., Gao, N. & Taylor, D.J. The process of mRNA-tRNA translocation. Proc. Natl. Acad. Sci. USA 104, 19671–19678 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Horan, L.H. & Noller, H.F. Intersubunit movement is required for ribosomal translocation. Proc. Natl. Acad. Sci. USA 104, 4881–4885 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Ermolenko, D.N. et al. The antibiotic viomycin traps the ribosome in an intermediate state of translocation. Nat. Struct. Mol. Biol. 14, 493–497 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Julián, P. et al. Structure of ratcheted ribosomes with tRNAs in hybrid states. Proc. Natl. Acad. Sci. USA 105, 16924–16927 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Cornish, P.V., Ermolenko, D.N., Noller, H.F. & Ha, T. Spontaneous intersubunit rotation in single ribosomes. Mol. Cell 30, 578–588 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Blanchard, S.C., Kim, H.D., Gonzalez, R.L. Jr., Puglisi, J.D. & Chu, S. tRNA dynamics on the ribosome during translation. Proc. Natl. Acad. Sci. USA 101, 12893–12898 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Ermolenko, D.N. et al. Observation of intersubunit movement of the ribosome in solution using FRET. J. Mol. Biol. 370, 530–540 (2007).

    Article  CAS  PubMed  Google Scholar 

  20. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Fei, J., Kosuri, P., MacDougall, D.D. & Gonzalez, R.L. Jr. Coupling of ribosomal L1 stalk and tRNA dynamics during translation elongation. Mol. Cell 30, 348–359 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Lill, R. et al. Specific recognition of the 3′-terminal adenosine of tRNAPhe in the exit site of Escherichia coli ribosomes. J. Mol. Biol. 203, 699–705 (1988).

    Article  CAS  PubMed  Google Scholar 

  24. Studer, S.M., Feinberg, J.S. & Joseph, S. Rapid kinetic analysis of EF-G-dependent mRNA translocation in the ribosome. J. Mol. Biol. 327, 369–381 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. Munro, J.B. et al. Spontaneous formation of the unlocked state of the ribosome is a multistep process. Proc. Natl. Acad. Sci. USA 107, 709–714 (2010).

    Article  CAS  PubMed  Google Scholar 

  26. Peske, F., Savelsbergh, A., Katunin, V.I., Rodnina, M.V. & Wintermeyer, W. Conformational changes of the small ribosomal subunit during elongation factor G-dependent tRNA-mRNA translocation. J. Mol. Biol. 343, 1183–1194 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Shi, X., Chiu, K., Ghosh, S. & Joseph, S. Bases in 16S rRNA important for subunit association, tRNA binding, and translocation. Biochemistry 48, 6772–6782 (2009).

    Article  CAS  PubMed  Google Scholar 

  28. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Borovinskaya, M.A., Shoji, S., Fredrick, K. & Cate, J.H. Structural basis for hygromycin B inhibition of protein biosynthesis. RNA 14, 1590–1599 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Borovinskaya, M.A., Shoji, S., Holton, J.M., Fredrick, K. & Cate, J.H. A steric block in translation caused by the antibiotic spectinomycin. ACS Chem. Biol. 2, 545–552 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zavialov, A.V., Hauryliuk, V.V. & Ehrenberg, M. Guanine-nucleotide exchange on ribosome-bound elongation factor G initiates the translocation of tRNAs. J. Biol. 4, 9 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Belitsina, N.V., Glukhova, M.A. & Spirin, A.S. Elongation factor G-promoted translocation and polypeptide elongation in ribosomes without GTP cleavage: use of columns with matrix-bound polyuridylic acid. Methods Enzymol. 60, 761–779 (1979).

    Article  CAS  PubMed  Google Scholar 

  33. Bodley, J.W., Zieve, F.J., Lin, L. & Zieve, S.T. Formation of the ribosome-G factor-GDP complex in the presence of fusidic acid. Biochem. Biophys. Res. Commun. 37, 437–443 (1969).

    Article  CAS  PubMed  Google Scholar 

  34. Inoue-Yokosawa, N., Ishikawa, C. & Kaziro, Y. The role of guanosine triphosphate in translocation reaction catalyzed by elongation factor G. J. Biol. Chem. 249, 4321–4323 (1974).

    CAS  PubMed  Google Scholar 

  35. Ticu, C., Nechifor, R., Nguyen, B., Desrosiers, M. & Wilson, K.S. Conformational changes in switch I of EF-G drive its directional cycling on and off the ribosome. EMBO J. 28, 2053–2065 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Savelsbergh, A., Rodnina, M.V. & Wintermeyer, W. Distinct functions of elongation factor G in ribosome recycling and translocation. RNA 15, 772–780 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 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).

    Article  CAS  PubMed  Google Scholar 

  38. Savelsbergh, A. et al. An elongation factor G-induced ribosome rearrangement precedes tRNA-mRNA translocation. Mol. Cell 11, 1517–1523 (2003).

    Article  CAS  PubMed  Google Scholar 

  39. Schuwirth, B.S. et al. Structures of the bacterial ribosome at 3.5 A resolution. Science 310, 827–834 (2005).

    Article  CAS  PubMed  Google Scholar 

  40. Ali, I.K., Lancaster, L., Feinberg, J., Joseph, S. & Noller, H.F. Deletion of a conserved, central ribosomal intersubunit RNA bridge. Mol. Cell 23, 865–874 (2006).

    Article  CAS  PubMed  Google Scholar 

  41. Feinberg, J.S. & Joseph, S. Ribose 2′-hydroxyl groups in the 5′ strand of the acceptor arm of P-site tRNA are not essential for EF-G catalyzed translocation. RNA 12, 580–588 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Feldman, M.B., Terry, D.S., Altman, R.B. & Blanchard, S.C. Aminoglycoside activity observed on single pre-translocation ribosome complexes. Nat. Chem. Biol. 6, 54–62 (2010).

    Article  CAS  PubMed  Google Scholar 

  43. Katunin, V.I., Savelsbergh, A., Rodnina, M.V. & Wintermeyer, W. Coupling of GTP hydrolysis by elongation factor G to translocation and factor recycling on the ribosome. Biochemistry 41, 12806–12812 (2002).

    Article  CAS  PubMed  Google Scholar 

  44. Robertson, J.M., Paulsen, H. & Wintermeyer, W. Pre-steady-state kinetics of ribosomal translocation. J. Mol. Biol. 192, 351–360 (1986).

    Article  CAS  PubMed  Google Scholar 

  45. Gao, Y.G. et al. The structure of the ribosome with elongation factor G trapped in the posttranslocational state. Science 326, 694–699 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Martemyanov, K.A., Yarunin, A.S., Liljas, A. & Gudkov, A.T. An intact conformation at the tip of elongation factor G domain IV is functionally important. FEBS Lett. 434, 205–208 (1998).

    Article  CAS  PubMed  Google Scholar 

  47. Peske, F., Matassova, N.B., Savelsbergh, A., Rodnina, M.V. & Wintermeyer, W. Conformationally restricted elongation factor G retains GTPase activity but is inactive in translocation on the ribosome. Mol. Cell 6, 501–505 (2000).

    Article  CAS  PubMed  Google Scholar 

  48. Wilson, K.S. & Noller, H.F. Mapping the position of translational elongation factor EF-G in the ribosome by directed hydroxyl radical probing. Cell 92, 131–139 (1998).

    Article  CAS  PubMed  Google Scholar 

  49. Culver, G.M. & Noller, H.F. Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins. RNA 5, 832–843 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Hickerson, R., Majumdar, Z.K., Baucom, A., Clegg, R.M. & Noller, H.F. Measurement of internal movements within the 30 S ribosomal subunit using Forster resonance energy transfer. J. Mol. Biol. 354, 459–472 (2005).

    Article  CAS  PubMed  Google Scholar 

  51. Lieberman, K.R. et al. The 23 S rRNA environment of ribosomal protein L9 in the 50 S ribosomal subunit. J. Mol. Biol. 297, 1129–1143 (2000).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

These studies were supported by grant no. GM-17129 from the US National Institutes of Health, US National Science Foundation (NSF) grant no. MCB-0723300 (to H.F.N.), and a North Atlantic Treaty Organization–NSF postdoctoral fellowship to D.N.E. The authors thank D. Herschlag, M. Rodnina, S. Joseph, A. Korostelev and L. Lancaster for critical discussions.

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  1. Dmitri N Ermolenko

    Present address: Present address: Department of Biochemistry and Biophysics and Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, New York, USA.,

Authors and Affiliations

  1. Center for Molecular Biology of RNA, University of California, Santa Cruz, Santa Cruz, California, USA.,

    Dmitri N Ermolenko & Harry F Noller

  2. Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, California, USA.,

    Dmitri N Ermolenko & Harry F Noller

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D.N.E. and H.F.N. designed the research; D.N.E. carried out the experiments; D.N.E. and H.F.N. analyzed the data and wrote the paper.

Corresponding author

Correspondence to Harry F Noller.

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Ermolenko, D., Noller, H. mRNA translocation occurs during the second step of ribosomal intersubunit rotation. Nat Struct Mol Biol 18, 457–462 (2011). https://doi.org/10.1038/nsmb.2011

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