Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Single-molecule FRET reveals the energy landscape of the full-length SAM-I riboswitch

Nature Chemical Biology volume 13, pages 1172–1178 (2017)Cite this article

Subjects

Abstract

S-adenosyl-L-methionine (SAM) ligand binding induces major structural changes in SAM-I riboswitches, through which gene expression is regulated via transcription termination. Little is known about the conformations and motions governing the function of the full-length Bacillus subtilis yitJ SAM-I riboswitch. Therefore, we have explored its conformational energy landscape as a function of Mg2+ and SAM ligand concentrations using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling analysis. We resolved four conformational states both in the presence and the absence of SAM and determined their Mg2+-dependent fractional populations and conformational dynamics, including state lifetimes, interconversion rate coefficients and equilibration timescales. Riboswitches with terminator and antiterminator folds coexist, and SAM binding only gradually shifts the populations toward terminator states. We observed a pronounced acceleration of conformational transitions upon SAM binding, which may be crucial for off-switching during the brief decision window before expression of the downstream gene.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structural depictions of the SAM-I riboswitch.
Figure 2: Mg2+- and ligand-dependent folding of SAM-I riboswitch.
Figure 3: Hidden Markov model (HMM) analysis of conformational dynamics of the SAM-I riboswitch.
Figure 4: Global fit analysis of smFRET efficiency histograms.
Figure 5: SAM-I riboswitch conformational states and their properties.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Montange, R.K. & Batey, R.T. Structure of the S-adenosylmethionine riboswitch regulatory mRNA element. Nature 441, 1172–1175 (2006).

    CAS  PubMed  Google Scholar 

  2. Winkler, W.C., Nahvi, A., Sudarsan, N., Barrick, J.E. & Breaker, R.R. An mRNA structure that controls gene expression by binding S-adenosylmethionine. Nat. Struct. Biol. 10, 701–707 (2003).

    CAS  PubMed  Google Scholar 

  3. Winkler, W.C. & Breaker, R.R. Genetic control by metabolite-binding riboswitches. ChemBioChem 4, 1024–1032 (2003).

    CAS  PubMed  Google Scholar 

  4. Montange, R.K. & Batey, R.T. Riboswitches: emerging themes in RNA structure and function. Annu. Rev. Biophys. 37, 117–133 (2008).

    CAS  PubMed  Google Scholar 

  5. Breaker, R.R. Prospects for riboswitch discovery and analysis. Mol. Cell 43, 867–879 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Tucker, B.J. & Breaker, R.R. Riboswitches as versatile gene control elements. Curr. Opin. Struct. Biol. 15, 342–348 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Wang, J.X. & Breaker, R.R. Riboswitches that sense S-adenosylmethionine and S-adenosylhomocysteine. Biochem. Cell Biol. 86, 157–168 (2008).

    CAS  PubMed  Google Scholar 

  8. Trausch, J.J. et al. Structural basis for diversity in the SAM clan of riboswitches. Proc. Natl. Acad. Sci. USA 111, 6624–6629 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. McDaniel, B.A., Grundy, F.J., Artsimovitch, I. & Henkin, T.M. Transcription termination control of the S box system: direct measurement of S-adenosylmethionine by the leader RNA. Proc. Natl. Acad. Sci. USA 100, 3083–3088 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Mustoe, A.M., Brooks, C.L. & Al-Hashimi, H.M. Hierarchy of RNA functional dynamics. Annu. Rev. Biochem. 83, 441–466 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Fürtig, B., Nozinovic, S., Reining, A. & Schwalbe, H. Multiple conformational states of riboswitches fine-tune gene regulation. Curr. Opin. Struct. Biol. 30, 112–124 (2015).

    PubMed  Google Scholar 

  12. Lemay, J.F., Penedo, J.C., Tremblay, R., Lilley, D.M. & Lafontaine, D.A. Folding of the adenine riboswitch. Chem. Biol. 13, 857–868 (2006).

    CAS  PubMed  Google Scholar 

  13. Greenleaf, W.J., Frieda, K.L., Foster, D.A., Woodside, M.T. & Block, S.M. Direct observation of hierarchical folding in single riboswitch aptamers. Science 319, 630–633 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Wickiser, J.K., Winkler, W.C., Breaker, R.R. & Crothers, D.M. The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch. Mol. Cell 18, 49–60 (2005).

    CAS  PubMed  Google Scholar 

  15. Neupane, K., Yu, H., Foster, D.A., Wang, F. & Woodside, M.T. Single-molecule force spectroscopy of the add adenine riboswitch relates folding to regulatory mechanism. Nucleic Acids Res. 39, 7677–7687 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Rieder, U., Kreutz, C. & Micura, R. Folding of a transcriptionally acting preQ1 riboswitch. Proc. Natl. Acad. Sci. USA 107, 10804–10809 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Stoddard, C.D. et al. Free state conformational sampling of the SAM-I riboswitch aptamer domain. Structure 18, 787–797 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Montange, R.K. et al. Discrimination between closely related cellular metabolites by the SAM-I riboswitch. J. Mol. Biol. 396, 761–772 (2010).

    CAS  PubMed  Google Scholar 

  19. Heppell, B. & Lafontaine, D.A. Folding of the SAM aptamer is determined by the formation of a K-turn-dependent pseudoknot. Biochemistry 47, 1490–1499 (2008).

    CAS  PubMed  Google Scholar 

  20. Heppell, B. et al. Molecular insights into the ligand-controlled organization of the SAM-I riboswitch. Nat. Chem. Biol. 7, 384–392 (2011).

    CAS  PubMed  Google Scholar 

  21. Klein, D.J., Schmeing, T.M., Moore, P.B. & Steitz, T.A. The kink-turn: a new RNA secondary structure motif. EMBO J. 20, 4214–4221 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Winkler, W.C., Grundy, F.J., Murphy, B.A. & Henkin, T.M. The GA motif: an RNA element common to bacterial antitermination systems, rRNA, and eukaryotic RNAs. RNA 7, 1165–1172 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. McDaniel, B.A., Grundy, F.J. & Henkin, T.M. A tertiary structural element in S box leader RNAs is required for S-adenosylmethionine-directed transcription termination. Mol. Microbiol. 57, 1008–1021 (2005).

    CAS  PubMed  Google Scholar 

  24. Lu, C. et al. SAM recognition and conformational switching mechanism in the Bacillus subtilis yitJ S box/SAM-I riboswitch. J. Mol. Biol. 404, 803–818 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Aboul-ela, F., Huang, W., Abd Elrahman, M., Boyapati, V. & Li, P. Linking aptamer-ligand binding and expression platform folding in riboswitches: prospects for mechanistic modeling and design. Wiley Interdiscip Rev RNA 6, 631–650 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Hennelly, S.P., Novikova, I.V. & Sanbonmatsu, K.Y. The expression platform and the aptamer: cooperativity between Mg2+ and ligand in the SAM-I riboswitch. Nucleic Acids Res. 41, 1922–1935 (2013).

    CAS  PubMed  Google Scholar 

  27. Boyapati, V.K., Huang, W., Spedale, J. & Aboul-Ela, F. Basis for ligand discrimination between ON and OFF state riboswitch conformations: the case of the SAM-I riboswitch. RNA 18, 1230–1243 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Frieda, K.L. & Block, S.M. Direct observation of cotranscriptional folding in an adenine riboswitch. Science 338, 397–400 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Kurschat, W.C., Müller, J., Wombacher, R. & Helm, M. Optimizing splinted ligation of highly structured small RNAs. RNA 11, 1909–1914 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Chung, H.S. & Gopich, I.V. Fast single-molecule FRET spectroscopy: theory and experiment. Phys. Chem. Chem. Phys. 16, 18644–18657 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Dammertz, K., Hengesbach, M., Helm, M., Nienhaus, G.U. & Kobitski, A.Y. Single-molecule FRET studies of counterion effects on the free energy landscape of human mitochondrial lysine tRNA. Biochemistry 50, 3107–3115 (2011).

    CAS  PubMed  Google Scholar 

  32. Kobitski, A.Y., Nierth, A., Helm, M., Jäschke, A. & Nienhaus, G.U. Mg2+-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis. Nucleic Acids Res. 35, 2047–2059 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Rieger, R., Kobitski, A., Sielaff, H. & Nienhaus, G.U. Evidence of a folding intermediate in RNase H from single-molecule FRET experiments. ChemPhysChem 12, 627–633 (2011).

    CAS  PubMed  Google Scholar 

  34. Hennelly, S.P. & Sanbonmatsu, K.Y. Tertiary contacts control switching of the SAM-I riboswitch. Nucleic Acids Res. 39, 2416–2431 (2011).

    CAS  PubMed  Google Scholar 

  35. Pirchi, M. et al. Single-molecule fluorescence spectroscopy maps the folding landscape of a large protein. Nat. Commun. 2, 493 (2011).

    PubMed  Google Scholar 

  36. McKinney, S.A., Joo, C. & Ha, T. Analysis of single-molecule FRET trajectories using hidden Markov modeling. Biophys. J. 91, 1941–1951 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Lee, T.H. Extracting kinetics information from single-molecule fluorescence resonance energy transfer data using hidden Markov models. J. Phys. Chem. B 113, 11535–11542 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Keller, B.G., Kobitski, A., Jäschke, A., Nienhaus, G.U. & Noé, F. Complex RNA folding kinetics revealed by single-molecule FRET and hidden Markov models. J. Am. Chem. Soc. 136, 4534–4543 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Prinz, J.H. et al. Markov models of molecular kinetics: generation and validation. J. Chem. Phys. 134, 174105 (2011).

    PubMed  Google Scholar 

  40. Huang, W., Kim, J., Jha, S. & Aboul-Ela, F. Conformational heterogeneity of the SAM-I riboswitch transcriptional ON state: a chaperone-like role for S-adenosyl methionine. J. Mol. Biol. 418, 331–349 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Huang, W., Kim, J., Jha, S. & Aboul-ela, F. The impact of a ligand binding on strand migration in the SAM-I riboswitch. PLoS Comput. Biol. 9, e1003069 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Reining, A. et al. Three-state mechanism couples ligand and temperature sensing in riboswitches. Nature 499, 355–359 (2013).

    CAS  PubMed  Google Scholar 

  43. Frauenfelder, H., Sligar, S.G. & Wolynes, P.G. The energy landscapes and motions of proteins. Science 254, 1598–1603 (1991).

    CAS  PubMed  Google Scholar 

  44. Nienhaus, G.U., Müller, J.D., McMahon, B.H. & Frauenfelder, H. Exploring the conformational energy landscape of proteins. Physica D 107, 297–311 (1997).

    CAS  Google Scholar 

  45. Baird, N.J., Kulshina, N. & Ferré-D'Amaré, A.R. Riboswitch function: flipping the switch or tuning the dimmer? RNA Biol. 7, 328–332 (2010).

    CAS  PubMed  Google Scholar 

  46. Suddala, K.C., Wang, J., Hou, Q. & Walter, N.G. Mg2+ shifts ligand-mediated folding of a riboswitch from induced-fit to conformational selection. J. Am. Chem. Soc. 137, 14075–14083 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Vogel, U. & Jensen, K.F. The RNA chain elongation rate in Escherichia coli depends on the growth rate. J. Bacteriol. 176, 2807–2813 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Huang, W., Kim, J., Jha, S. & Aboul-ela, F. A mechanism for S-adenosyl methionine assisted formation of a riboswitch conformation: a small molecule with a strong arm. Nucleic Acids Res. 37, 6528–6539 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Whitford, P.C. et al. Nonlocal helix formation is key to understanding S-adenosylmethionine-1 riboswitch function. Biophys. J. 96, L7–L9 (2009).

    PubMed  PubMed Central  Google Scholar 

  50. Heyes, C.D., Kobitski, A.Y., Amirgoulova, E.V. & Nienhaus, G.U. Biocompatible surfaces for specific tethering of individual protein molecules. J. Phys. Chem. B 108, 13387–13394 (2004).

    CAS  Google Scholar 

  51. Aitken, C.E., Marshall, R.A. & Puglisi, J.D. An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys. J. 94, 1826–1835 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Dave, R., Terry, D.S., Munro, J.B. & Blanchard, S.C. Mitigating unwanted photophysical processes for improved single-molecule fluorescence imaging. Biophys. J. 96, 2371–2381 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Kuzmenkina, E.V., Heyes, C.D. & Nienhaus, G.U. Single-molecule Forster resonance energy transfer study of protein dynamics under denaturing conditions. Proc. Natl. Acad. Sci. USA 102, 15471–15476 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Seyfried, V., Birk, H., Storz, R. & Ulrich, H. Advances in multispectral confocal imaging. Proc. SPIE 5139, 147–157 (2003).

    CAS  Google Scholar 

  55. Kapanidis, A.N. et al. Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules. Proc. Natl. Acad. Sci. USA 101, 8936–8941 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Osborne, M.A., Balasubramanian, S., Furey, W.S. & Klenerman, D. Optically biased diffusion of single molecules studied by confocal fluorescence microscopy. J. Phys. Chem. B 102, 3160–3167 (1998).

    CAS  Google Scholar 

  57. Saccà, B. et al. Reversible reconfiguration of DNA origami nanochambers monitored by single-molecule FRET. Angew. Chem. Int. Ed. Engl. 54, 3592–3597 (2015).

    PubMed  Google Scholar 

  58. Kobitski, A.Y., Hengesbach, M., Helm, M. & Nienhaus, G.U. Sculpting an RNA conformational energy landscape by a methyl group modification--a single-molecule FRET study. Angew. Chem. Int. Ed. Engl. 47, 4326–4330 (2008).

    CAS  PubMed  Google Scholar 

  59. Noé, F., Horenko, I., Schütte, C. & Smith, J.C. Hierarchical analysis of conformational dynamics in biomolecules: transition networks of metastable states. J. Chem. Phys. 126, 155102 (2007).

    PubMed  Google Scholar 

Download references

Acknowledgements

We thank A. Schug (KIT) for fruitful discussions. This work was supported by grants from the Karlsruhe Heidelberg Research Partnership (HEiKA) and the Volkswagen Foundation (grant 82549) to A.J. and G.U.N. G.U.N. was also funded by the Helmholtz program Science and Technology of Nanosystems (STN), Karlsruhe School of Optics and Photonics (KSOP) and the Deutsche Forschungsgemeinschaft (DFG), grant GRK 2039. B.G.K. was funded by the DFG through grant CRC 1114 (project B05).

Author information

Authors and Affiliations

  1. Institute of Applied Physics, Karlsruhe Institute of Technology, Karlsruhe, Germany

    Christoph Manz, Andrei Yu Kobitski & G Ulrich Nienhaus

  2. Heidelberg Karlsruhe Research Partnership (HEiKA), Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany

    Christoph Manz, Andres Jäschke & G Ulrich Nienhaus

  3. Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, Heidelberg, Germany

    Ayan Samanta & Andres Jäschke

  4. Freie Universität Berlin, Institute of Chemistry and Biochemistry, Berlin, Germany

    Bettina G Keller

  5. Institute of Nanotechnology and Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany

    G Ulrich Nienhaus

  6. Department of Physics, University of Illinois at Urbana–Champaign, Urbana, Illinois, USA

    G Ulrich Nienhaus

Authors
  1. Christoph Manz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrei Yu Kobitski
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ayan Samanta
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bettina G Keller
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andres Jäschke
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. G Ulrich Nienhaus
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.U.N. and A.J. designed research; A.S. synthesized RNA constructs; C.M. and A.Y.K. built the experimental setup and took data; C.M., A.Y.K. and B.G.K. analyzed data and all authors contributed to manuscript writing.

Corresponding author

Correspondence to G Ulrich Nienhaus.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1 and 2, and Supplementary Figures 1–8. (PDF 2215 kb)

Life Sciences Reporting Summary (PDF 129 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Manz, C., Kobitski, A., Samanta, A. et al. Single-molecule FRET reveals the energy landscape of the full-length SAM-I riboswitch. Nat Chem Biol 13, 1172–1178 (2017). https://doi.org/10.1038/nchembio.2476

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2476

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing