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Real-time observation of trigger factor function on translating ribosomes

Nature volume 444, pages 455–460 (2006)Cite this article

Abstract

The contribution of co-translational chaperone functions to protein folding is poorly understood. Ribosome-associated trigger factor (TF) is the first molecular chaperone encountered by nascent polypeptides in bacteria. Here we show, using fluorescence spectroscopy to monitor TF function and structural rearrangements in real time, that TF interacts with ribosomes and translating polypeptides in a dynamic reaction cycle. Ribosome binding stabilizes TF in an open, activated conformation. Activated TF departs from the ribosome after a mean residence time of ∼10 s, but may remain associated with the elongating nascent chain for up to 35 s, allowing entry of a new TF molecule at the ribosome docking site. The duration of nascent-chain interaction correlates with the occurrence of hydrophobic motifs in translating polypeptides, reflecting a high aggregation propensity. These findings can explain how TF prevents misfolding events during translation and may provide a paradigm for the regulation of nucleotide-independent chaperones.

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Figure 1: TF undergoes structural rearrangements upon binding to ribosomes.
Figure 2: TF remains associated with the nascent chain upon leaving the ribosome.
Figure 4: Chain-length dependence of TF interaction with titin I27 and model of TF function.
Figure 3: The nature of the nascent polypeptide influences its interaction with TF.

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References

  1. Frydman, J. Folding of newly translated proteins in vivo: the role of molecular chaperones. Annu. Rev. Biochem. 70, 603–647 (2001)

    Article  CAS  Google Scholar 

  2. Young, J. C., Agashe, V. R., Siegers, K. & Hartl, F. U. Pathways of chaperone-mediated protein folding in the cytosol. Nature Rev. Mol. Cell Biol. 5, 781–791 (2004)

    Article  CAS  Google Scholar 

  3. Deuerling, E. & Bukau, B. Chaperone-assisted folding of newly synthesized proteins in the cytosol. Crit. Rev. Biochem. Mol. Biol. 39, 261–277 (2004)

    Article  CAS  Google Scholar 

  4. Kramer, G. et al. L23 protein functions as a chaperone docking site on the ribosome. Nature 419, 171–174 (2002)

    Article  ADS  CAS  Google Scholar 

  5. Schlunzen, F. et al. The binding mode of the trigger factor on the ribosome: implications for protein folding and SRP interaction. Structure (Camb.) 13, 1685–1694 (2005)

    Article  Google Scholar 

  6. Baram, D. et al. Structure of trigger factor binding domain in biologically homologous complex with eubacterial ribosome reveals its chaperone action. Proc. Natl Acad. Sci. USA 102, 12017–12022 (2005)

    Article  ADS  CAS  Google Scholar 

  7. Hesterkamp, T., Hauser, S., Lutcke, H. & Bukau, B. Escherichia coli trigger factor is a prolyl isomerase that associates with nascent polypeptide chains. Proc. Natl Acad. Sci. USA 93, 4437–4441 (1996)

    Article  ADS  CAS  Google Scholar 

  8. Valent, Q. A. et al. Early events in preprotein recognition in E. coli: interaction of SRP and trigger factor with nascent polypeptides. EMBO J. 14, 5494–5505 (1995)

    Article  CAS  Google Scholar 

  9. Hartl, F. U. & Hayer-Hartl, M. Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295, 1852–1858 (2002)

    Article  ADS  CAS  Google Scholar 

  10. Agashe, V. R. et al. Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed. Cell 117, 199–209 (2004)

    Article  CAS  Google Scholar 

  11. Chang, H. C., Kaiser, C. M., Hartl, F. U. & Barral, J. M. De novo folding of GFP fusion proteins: high efficiency in eukaryotes but not in bacteria. J. Mol. Biol. 353, 397–409 (2005)

    Article  CAS  Google Scholar 

  12. Bremer, H. & Dennis, P. P. in Escherichia coli and Salmonella: Cellular and Molecular Biology (ed. Neidhart, F. C.) 1553–1569 (ASM Press, Washington DC, 1996)

  13. Lill, R., Crooke, E., Guthrie, B. & Wickner, W. The “trigger factor cycle” includes ribosomes, presecretory proteins, and the plasma membrane. Cell 54, 1013–1018 (1988)

    Article  CAS  Google Scholar 

  14. Maier, R., Eckert, B., Scholz, C., Lilie, H. & Schmid, F. X. Interaction of trigger factor with the ribosome. J. Mol. Biol. 326, 585–592 (2003)

    Article  CAS  Google Scholar 

  15. Ferbitz, L. et al. Trigger factor in complex with the ribosome forms a molecular cradle for nascent proteins. Nature 431, 590–596 (2004)

    Article  ADS  CAS  Google Scholar 

  16. Ludlam, A. V., Moore, B. A. & Xu, Z. The crystal structure of ribosomal chaperone trigger factor from Vibrio cholerae. Proc. Natl Acad. Sci. USA 101, 13436–13441 (2004)

    Article  ADS  CAS  Google Scholar 

  17. Genevaux, P. et al. In vivo analysis of the overlapping functions of DnaK and trigger factor. EMBO Rep. 5, 195–200 (2004)

    Article  CAS  Google Scholar 

  18. Kramer, G. et al. Functional dissection of Escherichia coli trigger factor: unraveling the function of individual domains. J. Bacteriol. 186, 3777–3784 (2004)

    Article  CAS  Google Scholar 

  19. Kramer, G. et al. Trigger factor peptidyl-prolyl cis/trans isomerase activity is not essential for the folding of cytosolic proteins in Escherichia coli.. J. Biol. Chem. 279, 14165–14170 (2004)

    Article  CAS  Google Scholar 

  20. Stryer, L. Fluorescence spectroscopy of proteins. Science 162, 526–533 (1968)

    Article  ADS  CAS  Google Scholar 

  21. Woolhead, C. A., McCormick, P. J. & Johnson, A. E. Nascent membrane and secretory proteins differ in FRET-detected folding far inside the ribosome and in their exposure to ribosomal proteins. Cell 116, 725–736 (2004)

    Article  CAS  Google Scholar 

  22. Schroder, G. F. & Grubmuller, H. FRETsg: biomolecular structure model building from multiple FRET experiments. Comp. Phys. Comm. 158, 150–157 (2004)

    Article  ADS  CAS  Google Scholar 

  23. Patzelt, H. et al. Three-state equilibrium of Escherichia coli trigger factor. Biol. Chem. 383, 1611–1619 (2002)

    Article  Google Scholar 

  24. Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nature Biotechnol. 19, 751–755 (2001)

    Article  CAS  Google Scholar 

  25. Patzelt, H. et al. Binding specificity of Escherichia coli trigger factor. Proc. Natl Acad. Sci. USA 98, 14244–14249 (2001)

    Article  ADS  CAS  Google Scholar 

  26. Chin, J. W., Martin, A. B., King, D. S., Wang, L. & Schultz, P. G. Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proc. Natl Acad. Sci. USA 99, 11020–11024 (2002)

    Article  ADS  CAS  Google Scholar 

  27. Michalski, C. J., Sells, B. H. & Morrison, M. Molecular morphology of ribosomes. Localization of ribosomal proteins in 50-S subunits. Eur. J. Biochem. 33, 481–485 (1973)

    Article  CAS  Google Scholar 

  28. Rudiger, S., Germeroth, L., Schneider-Mergener, J. & Bukau, B. Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. EMBO J. 16, 1501–1507 (1997)

    Article  CAS  Google Scholar 

  29. Roseman, M. A. Hydrophilicity of polar amino acid side-chains is markedly reduced by flanking peptide bonds. J. Mol. Biol. 200, 513–522 (1988)

    Article  CAS  Google Scholar 

  30. Creighton, T. E. Proteins: Structures and Molecular Properties (W. H. Freeman and Co., New York, 1984)

  31. Conti, E., Franks, N. P. & Brick, P. Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure 4, 287–298 (1996)

    Article  CAS  Google Scholar 

  32. Improta, S., Politou, A. S. & Pastore, A. Immunoglobulin-like modules from titin I-band: extensible components of muscle elasticity. Structure 4, 323–337 (1996)

    Article  CAS  Google Scholar 

  33. Marszalek, P. E. et al. Mechanical unfolding intermediates in titin modules. Nature 402, 100–103 (1999)

    Article  ADS  CAS  Google Scholar 

  34. Ban, N., Nissen, P., Hansen, J., Moore, P. B. & Steitz, T. A. The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science 289, 905–920 (2000)

    Article  ADS  CAS  Google Scholar 

  35. Harms, J. et al. High resolution structure of the large ribosomal subunit from a mesophilic eubacterium. Cell 107, 679–688 (2001)

    Article  CAS  Google Scholar 

  36. Ullers, R. S. et al. Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome. J. Cell Biol. 161, 679–684 (2003)

    Article  CAS  Google Scholar 

  37. Beck, K., Wu, L. F., Brunner, J. & Muller, M. Discrimination between SRP- and SecA/SecB-dependent substrates involves selective recognition of nascent chains by SRP and trigger factor. EMBO J. 19, 134–143 (2000)

    Article  CAS  Google Scholar 

  38. Eisner, G., Moser, M., Schafer, U., Beck, K. & Muller, M. Alternate recruitment of signal recognition particle and trigger factor to the signal sequence of a growing nascent polypeptide. J. Biol. Chem. 281, 7172–7179 (2006)

    Article  CAS  Google Scholar 

  39. Buskiewicz, I. et al. Trigger factor binds to ribosome-signal-recognition particle (SRP) complexes and is excluded by binding of the SRP receptor. Proc. Natl Acad. Sci. USA 101, 7902–7906 (2004)

    Article  ADS  CAS  Google Scholar 

  40. Raine, A., Ivanova, N., Wikberg, J. E. & Ehrenberg, M. Simultaneous binding of trigger factor and signal recognition particle to the E. coli ribosome. Biochimie 86, 495–500 (2004)

    Article  CAS  Google Scholar 

  41. Ullers, R. S. et al. Sequence-specific interactions of nascent Escherichia coli polypeptides with trigger factor and signal recognition particle. J. Biol. Chem. 281, 13999–14005 (2006)

    Article  CAS  Google Scholar 

  42. Chiti, F., Stefani, M., Taddei, N., Ramponi, G. & Dobson, C. M. Rationalization of the effects of mutations on peptide and protein aggregation rates. Nature 424, 805–808 (2003)

    Article  ADS  CAS  Google Scholar 

  43. Spedding, G. in Ribosomes and Protein Synthesis: A Practical Approach (ed. Spedding, G.) 1–29 (Oxford Univ. Press, Oxford, 1990)

  44. Kerner, M. J. et al. Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli.. Cell 122, 209–220 (2005)

    Article  CAS  Google Scholar 

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Acknowledgements

We thank A. Johnson for advice regarding assessment of fluorophore mobility, A. Bracher for help in designing the ΔPPIase construct and advice during building of the TF dimer model, M. Kerner for his help with bioinformatics analyses, R. Boteva and K. Chakroborty for discussion, P. Genevaux and C. Georgopoulos for the GP 367 E. coli strain and P. Schultz for reagents for in vivo crosslinking. J.M.B. was supported by a fellowship from the International Human Frontier Science Program Organization. Support by the Ernst Jung Foundation and the European Union is also acknowledged. Author Contributions F.U.H. was the project leader. C.M.K. and J.M.B. were responsible for experimental and project design. C.M.K. performed most of the experiments. V.R.A. made conceptual contributions and H.-C.C., V.R.A., S.K.L., S.A.E., M.H.-H. and J.M.B. performed some of the experiments. F.U.H. and J.M.B. wrote the manuscript.

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  1. Vishwas R. Agashe

    Present address: Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, OX3 9DS, UK

Authors and Affiliations

  1. Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152, Martinsried, Germany

    Christian M. Kaiser, Hung-Chun Chang, Vishwas R. Agashe, Sathish K. Lakshmipathy, Stephanie A. Etchells, Manajit Hayer-Hartl, F. Ulrich Hartl & José M. Barral

  2. Department of Neuroscience and Cell Biology, The University of Texas Medical Branch, 301 University Boulevard, Texas, 77555-0620, Galveston, USA

    José M. Barral

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Kaiser, C., Chang, HC., Agashe, V. et al. Real-time observation of trigger factor function on translating ribosomes. Nature 444, 455–460 (2006). https://doi.org/10.1038/nature05225

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