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The signal recognition particle contacts uL23 and scans substrate translation inside the ribosomal tunnel

Nature Microbiology volume 2, Article number: 16265 (2017) Cite this article

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Abstract

The signal recognition particle (SRP) delivers ∼25% of all bacterial proteins to the membrane for cotranslational insertion. However, a comprehensive model on how the low-abundant SRP scans the vast number of translating ribosomes to identify the correct substrates is lacking. Here, we show that the C-terminal helix of the signal-sequence-binding domain of SRP penetrates into the ribosomal tunnel and contacts the intra-tunnel loop of ribosomal protein uL23. This allows SRP to obtain information about the translational status of the ribosome and possibly the character of the approaching nascent chain. Correct substrates reposition the C-terminal helix of SRP, which facilitates stable binding of the signal sequence by the M-domain of SRP. Thus, SRP already surveys translating ribosomes before the signal sequence is surface exposed. This early interaction probably enables the small number of SRP molecules to scan many ribosomes and to initiate efficient targeting of proper substrates.

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Figure 1: Localization of uL23 at the E. coli ribosome.
Figure 2: Ffh contacts both the globular domain and the intra-tunnel loop of uL23.
Figure 3: The intra-tunnel loop of uL23 contacts the M-domain of Ffh.
Figure 4: Length-dependent contact of the intra-tunnel loop of uL23 with nascent membrane proteins.
Figure 5: Nascent membrane protein displaces SRP from the intra-tunnel loop.
Figure 6: Model for the early recognition steps during co-translational protein targeting in E. coli.

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References

  1. Denks, K. et al. The Sec translocon mediated protein transport in prokaryotes and eukaryotes. Mol. Membr. Biol. 31, 58–84 (2014).

    Article  CAS  Google Scholar 

  2. Bernstein, H. D. et al. Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle. Nature 340, 482–486 (1989).

    Article  CAS  Google Scholar 

  3. Powers, T. & Walter, P. Co-translational protein targeting catalyzed by the Escherichia coli signal recognition particle and its receptor. EMBO J. 16, 4880–4886 (1997).

    Article  CAS  Google Scholar 

  4. Gu, S.-Q., Peske, F., Wieden, H.-J., Rodnina, M. V. & Wintermeyer, W. The signal recognition particle binds to protein L23 at the peptide exit of the Escherichia coli ribosome. RNA 9, 566–573 (2003).

    Article  CAS  Google Scholar 

  5. Halic, M. et al. Structure of the signal recognition particle interacting with the elongation-arrested ribosome. Nature 427, 808–814 (2004).

    Article  CAS  Google Scholar 

  6. Schaffitzel, C. et al. Structure of the E. coli signal recognition particle bound to a translating ribosome. Nature 444, 503–506 (2006).

    Article  CAS  Google Scholar 

  7. Egea, P. F. et al. Substrate twinning activates the signal recognition particle and its receptor. Nature 427, 215–221 (2004).

    Article  CAS  Google Scholar 

  8. Focia, P. J., Shepotinovskaya, I. V., Seidler, J. A. & Freymann, D. M. Heterodimeric GTPase core of the SRP targeting complex. Science 303, 373–377 (2004).

    Article  CAS  Google Scholar 

  9. Keenan, R. J., Freymann, D. M., Walter, P. & Stroud, R. M. Crystal structure of the signal sequence binding subunit of the signal recognition particle. Cell 94, 181–191 (1998).

    Article  CAS  Google Scholar 

  10. Zopf, D., Bernstein, H. D., Johnson, A. E. & Walter, P. The methionine-rich domain of the 54 kD protein subunit of the signal recognition particle contains an RNA binding site and can be crosslinked to a signal sequence. EMBO J. 9, 4511–4517 (1990).

    Article  CAS  Google Scholar 

  11. Römisch, K., Webb, J., Lingelbach, K., Gausepohl, H. & Dobberstein, B. The 54-kD protein of signal recognition particle contains a methionine-rich RNA binding domain. J. Cell Biol. 111, 1793–1802 (1990).

    Article  Google Scholar 

  12. Rapiejko, P. J. & Gilmore, R. Signal sequence recognition and targeting of ribosomes to the endoplasmic reticulum by the signal recognition particle do not require GTP. Mol. Biol. Cell 5, 887–897 (1994).

    Article  CAS  Google Scholar 

  13. Zhang, X., Rashid, R., Wang, K. & Shan, S. Sequential checkpoints govern substrate selection during cotranslational protein targeting. Science 328, 757–760 (2010).

    Article  CAS  Google Scholar 

  14. Braig, D. et al. Signal sequence-independent SRP-SR complex formation at the membrane suggests an alternative targeting pathway within the SRP cycle. Mol. Biol. Cell 22, 2309–2323 (2011).

    Article  CAS  Google Scholar 

  15. Holtkamp, W. et al. Dynamic switch of the signal recognition particle from scanning to targeting. Nat. Struct. Mol. Biol. 19, 1332–1337 (2012).

    Article  CAS  Google Scholar 

  16. Shen, K., Arslan, S., Akopian, D., Ha, T. & Shan, S. Activated GTPase movement on an RNA scaffold drives co-translational protein targeting. Nature 492, 271–275 (2012).

    Article  CAS  Google Scholar 

  17. Saraogi, I., Akopian, D. & Shan, S.-O. Regulation of cargo recognition, commitment, and unloading drives cotranslational protein targeting. J. Cell Biol. 205, 693–706 (2014).

    Article  CAS  Google Scholar 

  18. von Loeffelholz, O. et al. Ribosome-SRP-FtsY cotranslational targeting complex in the closed state. Proc. Natl Acad. Sci. USA 112, 3943–3948 (2015).

    Article  CAS  Google Scholar 

  19. Kuhn, P. et al. Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon. J. Cell Biol. 211, 91–104 (2015).

    Article  CAS  Google Scholar 

  20. Jomaa, A., Boehringer, D., Leibundgut, M. & Ban, N. Structures of the E. coli translating ribosome with SRP and its receptor and with the translocon. Nat. Commun. 7, 10471 (2016).

    Article  CAS  Google Scholar 

  21. Siegel, V. & Walter, P. The affinity of signal recognition particle for presecretory proteins is dependent on nascent chain length. EMBO J. 7, 1769–1775 (1988).

    Article  CAS  Google Scholar 

  22. Noriega, T. R., Chen, J., Walter, P. & Puglisi, J. D. Real-time observation of signal recognition particle binding to actively translating ribosomes. eLife 3, e04418 (2014).

    Article  Google Scholar 

  23. Bornemann, T., Jöckel, J., Rodnina, M. V. & Wintermeyer, W. Signal sequence-independent membrane targeting of ribosomes containing short nascent peptides within the exit tunnel. Nat. Struct. Mol. Biol. 15, 494–499 (2008).

    Article  CAS  Google Scholar 

  24. Berndt, U., Oellerer, S., Zhang, Y., Johnson, A. E. & Rospert, S. A signal-anchor sequence stimulates signal recognition particle binding to ribosomes from inside the exit tunnel. Proc. Natl Acad. Sci. USA 106, 1398–1403 (2009).

    Article  CAS  Google Scholar 

  25. Voorhees, R. M. & Hegde, R. S. Structures of the scanning and engaged states of the mammalian SRP–ribosome complex. eLife 4, e07975 (2015).

    Article  Google Scholar 

  26. 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 

  27. Pool, M. R., Stumm, J., Fulga, T. A., Sinning, I. & Dobberstein, B. Distinct modes of signal recognition particle interaction with the ribosome. Science 297, 1345–1348 (2002).

    Article  CAS  Google Scholar 

  28. Ryu, Y. & Schultz, P. G. Efficient incorporation of unnatural amino acids into proteins in Escherichia coli. Nat. Methods 3, 263–265 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  30. Huber, D. et al. SecA interacts with ribosomes in order to facilitate posttranslational translocation in bacteria. Mol. Cell 41, 343–353 (2011).

    Article  CAS  Google Scholar 

  31. Halic, M. et al. Following the signal sequence from ribosomal tunnel exit to signal recognition particle. Nature 444, 507–511 (2006).

    Article  CAS  Google Scholar 

  32. Sachelaru, I. et al. YidC occupies the lateral gate of the SecYEG translocon and is sequentially displaced by a nascent membrane protein. J. Biol. Chem. 288, 16295–16307 (2013).

    Article  CAS  Google Scholar 

  33. Giglione, C., Fieulaine, S. & Meinnel, T. N-terminal protein modifications bringing back into play the ribosome. Biochimie 114, 134–146 (2015).

    Article  CAS  Google Scholar 

  34. Lütcke, H., High, S., Römisch, K., Ashford, A. J. & Dobberstein, B. The methionine-rich domain of the 54 kDa subunit of signal recognition particle is sufficient for the interaction with signal sequences. EMBO J. 11, 1543–1551 (1992).

    Article  Google Scholar 

  35. Hainzl, T., Huang, S., Meriläinen, G., Brännström, K. & Sauer-Eriksson, A. E. Structural basis of signal-sequence recognition by the signal recognition particle. Nat. Struct. Mol. Biol. 18, 389–391 (2011).

    Article  CAS  Google Scholar 

  36. Hainzl, T. & Sauer-Eriksson, A. E. Signal-sequence induced conformational changes in the signal recognition particle. Nat. Commun. 6, 7163 (2015).

    Article  CAS  Google Scholar 

  37. 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 

  38. Houben, E. N. G., Zarivach, R., Oudega, B. & Luirink, J. Early encounters of a nascent membrane protein specificity and timing of contacts inside and outside the ribosome. J. Cell Biol. 170, 27–35 (2005).

    Article  CAS  Google Scholar 

  39. 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  CAS  Google Scholar 

  40. Lee, J. I., Kuhn, A. & Dalbey, R. E. Distinct domains of an oligotopic membrane protein are Sec-dependent and Sec-independent for membrane insertion. J. Biol. Chem. 267, 938–943 (1992).

    CAS  PubMed  Google Scholar 

  41. Scotti, P. A. et al. SecA is not required for signal recognition particle-mediated targeting and initial membrane insertion of a nascent inner membrane protein. J. Biol. Chem. 274, 29883–29888 (1999).

    Article  CAS  Google Scholar 

  42. Noriega, T. R. et al. Signal recognition particle–ribosome binding is sensitive to nascent chain length. J. Biol. Chem. 289, 19294–19305 (2014).

    Article  CAS  Google Scholar 

  43. Flanagan, J. J. et al. Signal recognition particle binds to ribosome-bound signal sequences with fluorescence-detected subnanomolar affinity that does not diminish as the nascent chain lengthens. J. Biol. Chem. 278, 18628–18637 (2003).

    Article  CAS  Google Scholar 

  44. Schibich, D. et al. Global profiling of SRP interaction with nascent polypeptides. Nature 536, 219–223 (2016).

    Article  CAS  Google Scholar 

  45. Sandikci, A. et al. Dynamic enzyme docking to the ribosome coordinates N-terminal processing with polypeptide folding. Nat. Struct. Mol. Biol. 20, 843–850 (2013).

    Article  CAS  Google Scholar 

  46. Nyathi, Y. & Pool, M. R. Analysis of the interplay of protein biogenesis factors at the ribosome exit site reveals new role for NAC. J. Cell Biol. 210, 287–301 (2015).

    Article  CAS  Google Scholar 

  47. Siegel, V. & Walter, P. Elongation arrest is not a prerequisite for secretory protein translocation across the microsomal membrane. J. Cell Biol. 100, 1913–1921 (1985).

    Article  CAS  Google Scholar 

  48. 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  CAS  Google Scholar 

  49. Nilsson, O. B. et al. Cotranslational protein folding inside the ribosome exit tunnel. Cell Rep. 12, 1533–1540 (2015).

    Article  CAS  Google Scholar 

  50. Hanahan, D. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557–580 (1983).

    Article  CAS  Google Scholar 

  51. Deitermann, S., Sprie, G. S. & Koch, H.-G. A dual function for SecA in the assembly of single spanning membrane proteins in Escherichia coli. J. Biol. Chem. 280, 39077–39085 (2005).

    Article  CAS  Google Scholar 

  52. Behrmann, M. et al. Requirements for the translocation of elongation-arrested, ribosome-associated OmpA across the plasma membrane of Escherichia coli. J. Biol. Chem. 273, 13898–13904 (1998).

    Article  CAS  Google Scholar 

  53. Yosef, I., Bochkareva, E. S. & Bibi, E. Escherichia coli SRP, its protein subunit Ffh, and the Ffh M domain are able to selectively limit membrane protein expression when overexpressed. mBio 1, e00020-10 (2010).

    Article  Google Scholar 

  54. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    Article  CAS  Google Scholar 

  55. Giacalone, M. J. et al. Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system. Biotechniques 40, 355–364 (2006).

    Article  CAS  Google Scholar 

  56. Keen, N. T., Tamaki, S., Kobayashi, D. & Trollinger, D. Improved broad-host-range plasmids for DNA cloning in Gram-negative bacteria. Gene 70, 191–197 (1988).

    Article  CAS  Google Scholar 

  57. Yap, M.-N. & Bernstein, H. D. The plasticity of a translation arrest motif yields insights into nascent polypeptide recognition inside the ribosome tunnel. Mol. Cell 34, 201–211 (2009).

    Article  CAS  Google Scholar 

  58. Schaffitzel, C. & Ban, N. Generation of ribosome nascent chain complexes for structural and functional studies. J. Struct. Biol. 158, 463–471 (2007).

    Article  CAS  Google Scholar 

  59. Koch, H. G. et al. In vitro studies with purified components reveal signal recognition particle (SRP) and SecA/SecB as constituents of two independent protein-targeting pathways of Escherichia coli. Mol. Biol. Cell 10, 2163–2173 (1999).

    Article  CAS  Google Scholar 

  60. Bischoff, L., Wickles, S., Berninghausen, O., van der Sluis, E. O. & Beckmann, R. Visualization of a polytopic membrane protein during SecY-mediated membrane insertion. Nat. Commun. 5, 4103 (2014).

    Article  CAS  Google Scholar 

  61. Welte, T. et al. Promiscuous targeting of polytopic membrane proteins to SecYEG or YidC by the Escherichia coli signal recognition particle. Mol. Biol. Cell 23, 464–479 (2012).

    Article  CAS  Google Scholar 

  62. Müller, M. & Blobel, G. In vitro translocation of bacterial proteins across the plasma membrane of Escherichia coli. Proc. Natl Acad. Sci. USA 81, 7421–7425 (1984).

    Article  Google Scholar 

  63. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH image to ImageJ 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    Article  CAS  Google Scholar 

  64. Beck, K., Wu, L. F., Brunner, J. & Müller, 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 

  65. Hanes, J. & Plückthun, A. In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl Acad. Sci. USA 94, 4937–4942 (1997).

    Article  CAS  Google Scholar 

  66. Valent, Q. A. et al. Nascent membrane and presecretory proteins synthesized in Escherichia coli associate with signal recognition particle and trigger factor. Mol. Microbiol. 25, 53–64 (1997).

    Article  CAS  Google Scholar 

  67. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

  68. Noeske, J. et al. High-resolution structure of the Escherichia coli ribosome. Nat. Struct. Mol. Biol. 22, 336–341 (2015).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank G. Kramer (University Heidelberg) for providing the rplW deletion strain, T. Bornemann and W. Wintermeyer (MPI Biophysical Chemistry, Göttingen) for plasmid pCDF-L23 and purified MAP, PDF and TF, E. Bibi (Weizmann Institute, Israel) for plasmids pIY1043 and pIY1045, D. Vikström and G. von Heijne (University Stockholm) for plasmid pRha and the LepB construct, and C. Schaffitzel (University Bristol) for the FtsQ construct. The authors also thank B. Graf for the automated cell size measurement protocol. This work was supported by grants from the DFG (FOR967, FOR929, GRK2202 and KO2184/8) and by a ‘Fill-in-the-gap’ fellowship of the University Freiburg Medical School.

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  1. Institut für Biochemie und Molekularbiologie, ZBMZ, Faculty of Medicine, Albert-Ludwigs Universität Freiburg, Stefan Meier Strasse 17, 79104 Freiburg, Germany

    Kärt Denks, Nadine Sliwinski, Veronika Erichsen, Bogdana Borodkina, Andrea Origi & Hans-Georg Koch

  2. Faculty of Biology, Albert-Ludwigs Universität Freiburg, Stefan Meier Strasse 17, 79104 Freiburg, Germany

    Kärt Denks & Andrea Origi

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  1. Kärt Denks
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K.D. carried out study design, experimental work, data analyses and writing of the manuscript. N.S., V.E., B.B. and A.O. performed experimental work and data analyses. H.-G.K. carried out study design and data analyses and contributed to writing the manuscript.

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Correspondence to Hans-Georg Koch.

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Supplementary Figures 1–7, Supplementary Notes and Discussion, Supplementary References. (PDF 4646 kb)

Supplementary Table 1

Oligonucleotide list. (XLS 37 kb)

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Denks, K., Sliwinski, N., Erichsen, V. et al. The signal recognition particle contacts uL23 and scans substrate translation inside the ribosomal tunnel. Nat Microbiol 2, 16265 (2017). https://doi.org/10.1038/nmicrobiol.2016.265

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