Article | Published:

A conserved proline switch on the ribosome facilitates the recruitment and binding of trGTPases

Nature Structural & Molecular Biology volume 19, pages 403410 (2012) | Download Citation

Abstract

When elongation factor G (EF-G) binds to the ribosome, it first makes contact with the C-terminal domain (CTD) of L12 before interacting with the N-terminal domain (NTD) of L11. Here we have identified a universally conserved residue, Pro22 of L11, that functions as a proline switch (PS22), as well as the corresponding center of peptidyl-prolyl cis-trans isomerase (PPIase) activity on EF-G that drives the cis-trans isomerization of PS22. Only the cis configuration of PS22 allows direct contact between the L11 NTD and the L12 CTD. Mutational analyses of both PS22 and the residues of the EF-G PPIase center reveal their function in translational GTPase (trGTPase) activity, protein synthesis and cell survival in Escherichia coli. Finally, we demonstrate that all known universal trGTPases contain an active PPIase center. Our observations suggest that the cis-trans isomerization of the L11 PS22 is a universal event required for an efficient turnover of trGTPases throughout the translation process.

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References

  1. 1.

    et al. Structural basis for the function of the ribosomal L7/12 stalk in factor binding and GTPase activation. Cell 121, 991–1004 (2005).

  2. 2.

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

  3. 3.

    et al. Structural basis for interaction of the ribosome with the switch regions of GTP-bound elongation factors. Mol. Cell 25, 751–764 (2007).

  4. 4.

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

  5. 5.

    et al. Conformation and dynamics of ribosomal stalk protein L12 in solution and on the ribosome. Biochemistry 43, 5930–5936 (2004).

  6. 6.

    et al. The ribosomal stalk binds to translation factors IF2, EF-Tu, EF-G and RF3 via a conserved region of the L12 C-terminal domain. J. Mol. Biol. 365, 468–479 (2007).

  7. 7.

    , , , & Interaction of the G′ domain of elongation factor G and the C-terminal domain of ribosomal protein L7/L12 during translocation as revealed by cryo-EM. Mol. Cell 20, 723–731 (2005).

  8. 8.

    , & Functional interactions between the G' subdomain of bacterial translation factor EF-G and ribosomal protein L7/L12. J. Biol. Chem. 282, 36998–37005 (2007).

  9. 9.

    , , , & Control of phosphate release from elongation factor G by ribosomal protein L7/12. EMBO J. 24, 4316–4323 (2005).

  10. 10.

    , , & Guanosinetriphosphatase activity dependent on elongation factor Tu and ribosomal protein L7/L12. Proc. Natl. Acad. Sci. USA 75, 3192–3195 (1978).

  11. 11.

    , & GTPase activation of elongation factors Tu and G on the ribosome. Biochemistry 41, 12520–12528 (2002).

  12. 12.

    & Concerning the mode of action of micrococcin upon bacterial protein synthesis. Eur. J. Biochem. 118, 47–52 (1981).

  13. 13.

    Thiostrepton: a ribosomal inhibitor of translocation. Biochem. Biophys. Res. Commun. 40, 667–674 (1970).

  14. 14.

    , , , & L11 domain rearrangement upon binding to RNA and thiostrepton studied by NMR spectroscopy. Nucleic Acids Res. 35, 441–454 (2007).

  15. 15.

    et al. Thiostrepton inhibits the turnover but not the GTPase of elongation factor G on the ribosome. Proc. Natl. Acad. Sci. USA 96, 9586–9590 (1999).

  16. 16.

    et al. Translational regulation via L11: molecular switches on the ribosome turned on and off by thiostrepton and micrococcin. Mol. Cell 30, 26–38 (2008).

  17. 17.

    & Structure of the base of the L7/L12 stalk of the Haloarcula marismortui large ribosomal subunit: analysis of L11 movements. J. Mol. Biol. 371, 1047–1059 (2007).

  18. 18.

    , , & Functional conformations of the L11-ribosomal RNA complex revealed by correlative analysis of cryo-EM and molecular dynamics simulations. RNA 12, 1240–1253 (2006).

  19. 19.

    et al. Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites. Nature 468, 713–716 (2010).

  20. 20.

    , , , & A detailed view of a ribosomal active site: The structure of the L11-RNA complex. Cell 97, 491–502 (1999).

  21. 21.

    , , & Structural aspects of messenger RNA reading frame maintenance by the ribosome. Nat. Struct. Mol. Biol. 17, 555–560 (2010).

  22. 22.

    , , & Structures of the Escherichia coli ribosome with antibiotics bound near the peptidyl transferase center explain spectra of drug action. Proc. Natl. Acad. Sci. USA 107, 17152–17157 (2010).

  23. 23.

    , , & Structure of the 70S ribosome bound to release factor 2 and a substrate analog provides insights into catalysis of peptide release. Proc. Natl. Acad. Sci. USA 107, 8593–8598 (2010).

  24. 24.

    , , , & The cryo-EM structure of a translation initiation complex from Escherichia coli. Cell 121, 703–712 (2005).

  25. 25.

    et al. GTPase activation of elongation factor EF-Tu by the ribosome during decoding. EMBO J. 28, 755–765 (2009).

  26. 26.

    et al. A new tRNA intermediate revealed on the ribosome during EF4-mediated back-translocation. Nat. Struct. Mol. Biol. 15, 910–915 (2008).

  27. 27.

    et al. RF3 induces ribosomal conformational changes responsible for dissociation of class I release factors. Cell 129, 929–941 (2007).

  28. 28.

    & The X-Pro peptide bond as an nmr probe for conformational studies of flexible linear peptides. Biopolymers 15, 2025–2041 (1976).

  29. 29.

    & Insights into the catalytic mechanism of peptidyl prolyl cis/trans isomerases. Front. Biosci. 9, 3453–3478 (2004).

  30. 30.

    , , & 1H, 15N, and 13C assignments and secondary structure identification for full-length ribosomal protein L11 from Thermus thermophilus. J. Biomol. NMR 20, 293–294 (2001).

  31. 31.

    , , & Acid denaturation and refolding of green fluorescent protein. Biochemistry 43, 14238–14248 (2004).

  32. 32.

    & Trigger factor assisted folding of green fluorescent protein. Biochemistry 47, 348–357 (2008).

  33. 33.

    & Trigger factor-assisted folding of bovine carbonic anhydrase II. Biochem. Biophys. Res. Commun. 313, 509–515 (2004).

  34. 34.

    , , , & Mip protein of Legionella pneumophila exhibits peptidyl-prolyl-cis/trans isomerase (PPlase) activity. Mol. Microbiol. 6, 1375–1383 (1992).

  35. 35.

    , , & Crystal structure of the eukaryotic ribosome. Science 330, 1203–1209 (2010).

  36. 36.

    , , & Visualization of elongation factor G on the Escherichia coli 70S ribosome: The mechanism of translocation. Proc. Natl. Acad. Sci. USA 95, 6134–6138 (1998).

  37. 37.

    , , , & Domain motions of EF-G bound to the 70S ribosome: Insights from a hand-shaking between multi-resolution structures. Biophys. J. 79, 1670–1678 (2000).

  38. 38.

    , , , & The process of mRNA-tRNA translocation. Proc. Natl. Acad. Sci. USA 104, 19671–19678 (2007).

  39. 39.

    , , & Molecular dynamics of EF-G during translocation. Proteins 79, 1478–1486 (2011).

  40. 40.

    The ribosome as a conveying thermal ratchet machine. J. Biol. Chem. 284, 21103–21119 (2009).

  41. 41.

    et al. Structural insights into cognate versus near-cognate discrimination during decoding. EMBO J. 30, 1497–1507 (2011).

  42. 42.

    , , & The mechanism for activation of GTP hydrolysis on the ribosome. Science 330, 835–838 (2010).

  43. 43.

    et al. The crystal structure of the ribosome bound to EF-Tu and aminoacyl-tRNA. Science 326, 688–694 (2009).

  44. 44.

    , , & The structure of LepA, the ribosomal back translocase. Proc. Natl. Acad. Sci. USA 105, 4673–4678 (2008).

  45. 45.

    , , , & Localization of L11 protein on the ribosome and elucidation of its involvement in EF-G-dependent translocation. J. Mol. Biol. 311, 777–787 (2001).

  46. 46.

    , , & Purification and properties of Escherichia coli translational initiation factors. Biochem. Int. 2, 421–428 (1981).

  47. 47.

    et al. Dissecting the ribosomal inhibition mechanisms of edeine and pactamycin: the universally conserved residues G693 and C795 regulate P-site RNA binding. Mol. Cell 13, 113–124 (2004).

  48. 48.

    , & The ribosomal binding and peptidyl-tRNA hydrolysis functions of Escherichia coli release factor 2 are linked through residue 246. RNA 6, 1704–1713 (2000).

  49. 49.

    et al. A ribosome-associated peptidyl-prolyl cis/trans isomerase identified as the trigger factor. EMBO J. 14, 4939–4948 (1995).

  50. 50.

    , , & Reassessment of the putative chaperone function of prolyl-cis/trans-isomerases. FEBS Lett. 348, 145–148 (1994).

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Acknowledgements

We are very grateful to J. Frank and Y. Hashem for their critical discussions and great efforts in polishing the manuscript. We thank J. Wang, J. Zhou, J. Lin, Y. Tao and J. Lou for their technical support and help. We thank Y. Feng for NMR analysis, G. Liu and J. Xue for preparing some of the proteins and W. Zhang for creating the movie. This work was supported by grants from the Major State Basic Research Development Program of China (2012CB911001 and 2010CB834201), the National Natural Science Foundation of China (31170756) and the Novo Nordisk-Chinese Academy of Sciences Research Foundation.

Author information

Affiliations

  1. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.

    • Li Wang
    • , Fang Yang
    • , Dejiu Zhang
    • , Zhi Chen
    • , Rui-Ming Xu
    • , Weimin Gong
    •  & Yan Qin
  2. School of Life Sciences, University of Science and Technology of China, Hefei, China.

    • Li Wang
    • , Weimin Gong
    •  & Yan Qin
  3. School of Life Sciences, University of Science and Technology of China, Hefei, China.

    • Knud H Nierhaus

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Contributions

Y.Q., K.H.N., W.G. and R.-M.X. designed the experiments. L.W., F.Y. and D.Z. conducted the experiments. Z.C. developed the molecular dynamics simulation. Y.Q. and L.W. analyzed the data. Y.Q., L.W. and K.H.N. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Yan Qin.

Supplementary information

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    Supplementary Text and Figures

    Supplementary Figures 1–3, Supplementary Tables 1–6 and Supplementary Methods

Videos

  1. 1.

    Supplementary Movie

    The Switch-and-Latch model. The movie demonstrates the conformational change of L11–L12-CTD complex between the open and the closed states. Open state (L11 in magenta, L12 in yellow, the proline switch trans-PS22 in green and loop 62 in cyan) is represented by the L11 on the ribosome•RF2 complex (PDB 2X9S, chain K) and L12-CTD in its isolated form (PDB 1CTF). Closed state (L11 in red, L12 in yellow, cis-PS22 in green and loop 62 in cyan) is presented by the L11 on the ribosome•EF-G•GDP•fusidic acid complex (PDB 2WRL, chain K) and L12 from the same complex (PDB 2WRL, chain L). The model was built based on MD simulation of L11-NTD. The animation is generated by the Morph Server and PyMol.

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DOI

https://doi.org/10.1038/nsmb.2254

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