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 analysis reveals multi-state folding of a guanine riboswitch

Nature Chemical Biology volume 13, pages 194–201 (2017)Cite this article

Subjects

Abstract

Guanine-responsive riboswitches undergo ligand-dependent structural rearrangements to control gene expression by transcription termination. While the molecular basis for ligand recognition is well established, the associated structural rearrangements and the kinetics involved in the formation of the aptamer domain are less well understood. Using high-resolution optical tweezers, we followed the folding trajectories of a single molecule of the xpt–pbuX guanine aptamer from Bacillus subtilis. We report a rapid six-state conformational rearrangement, in which three of the states are guanine dependent, during the transition from the linear to the native receptor conformation. The folding completes in <1 s. The force-dependent equilibrium kinetics and the mutational data indicated that the flexible J2–J3 junction undergoes a ligand-dependent conformational switching, which triggers the formation of the long-range tertiary interactions and the P1 helix. In the absence of the right ligand, the junction failed to initiate the series of conformational rearrangements required for the riboswitch activities.

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: The G-aptamer adopts a flexible conformation in the absence of ligand.
Figure 2: The folding pathways and the kinetics for the G-aptamer.
Figure 3: Kissing-loop mutations adversely affect ligand binding.
Figure 4: Rate constants for the structural elements in the wt and mutant aptamers.
Figure 5: Kissing-loop dynamics control transcription in purine riboswitches.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Mandal, M., Boese, B., Barrick, J.E., Winkler, W.C. & Breaker, R.R. Riboswitches control fundamental biochemical pathways in Bacillus subtilis and other bacteria. Cell 113, 577–586 (2003).

    Article  CAS  Google Scholar 

  2. Mandal, M. & Breaker, R.R. Adenine riboswitches and gene activation by disruption of a transcription terminator. Nat. Struct. Mol. Biol. 11, 29–35 (2004).

    Article  CAS  Google Scholar 

  3. Batey, R.T., Gilbert, S.D. & Montange, R.K. Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine. Nature 432, 411–415 (2004).

    Article  CAS  Google Scholar 

  4. Serganov, A. et al. Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs. Chem. Biol. 11, 1729–1741 (2004).

    Article  CAS  Google Scholar 

  5. Noeske, J. et al. An intermolecular base triple as the basis of ligand specificity and affinity in the guanine- and adenine-sensing riboswitch RNAs. Proc. Natl. Acad. Sci. USA 102, 1372–1377 (2005).

    Article  CAS  Google Scholar 

  6. Batey, R.T. Structure and mechanism of purine-binding riboswitches. Q. Rev. Biophys. 45, 345–381 (2012).

    Article  CAS  Google Scholar 

  7. Serganov, A. & Patel, D.J. Molecular recognition and function of riboswitches. Curr. Opin. Struct. Biol. 22, 279–286 (2012).

    Article  CAS  Google Scholar 

  8. Buck, J., Fürtig, B., Noeske, J., Wöhnert, J. & Schwalbe, H. Time-resolved NMR methods resolving ligand-induced RNA folding at atomic resolution. Proc. Natl. Acad. Sci. USA 104, 15699–15704 (2007).

    Article  CAS  Google Scholar 

  9. Lee, M.-K., Gal, M., Frydman, L. & Varani, G. Real-time multidimensional NMR follows RNA folding with second resolution. Proc. Natl. Acad. Sci. USA 107, 9192–9197 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Rieder, R., Lang, K., Graber, D. & Micura, R. Ligand-induced folding of the adenosine deaminase A-riboswitch and implications on riboswitch translational control. ChemBioChem 8, 896–902 (2007).

    Article  CAS  Google Scholar 

  12. Brenner, M.D., Scanlan, M.S., Nahas, M.K., Ha, T. & Silverman, S.K. Multivector fluorescence analysis of the xpt guanine riboswitch aptamer domain and the conformational role of guanine. Biochemistry 49, 1596–1605 (2010).

    Article  CAS  Google Scholar 

  13. St-Pierre, P., McCluskey, K., Shaw, E., Penedo, J.C. & Lafontaine, D.A. Fluorescence tools to investigate riboswitch structural dynamics. Biochim. Biophys. Acta 1839, 1005–1019 (2014).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. 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).

    Article  CAS  Google Scholar 

  17. Savinov, A., Perez, C.F. & Block, S.M. Single-molecule studies of riboswitch folding. Biochim. Biophys. Acta 1839, 1030–1045 (2014).

    Article  CAS  Google Scholar 

  18. Smith, S.B., Cui, Y. & Bustamante, C. Optical-trap force transducer that operates by direct measurement of light momentum. Methods Enzymol. 361, 134–162 (2003).

    Article  CAS  Google Scholar 

  19. Buck, J., Noeske, J., Wöhnert, J. & Schwalbe, H. Dissecting the influence of Mg2+ on 3D architecture and ligand-binding of the guanine-sensing riboswitch aptamer domain. Nucleic Acids Res. 38, 4143–4153 (2010).

    Article  CAS  Google Scholar 

  20. Ottink, O.M. et al. Ligand-induced folding of the guanine-sensing riboswitch is controlled by a combined predetermined induced fit mechanism. RNA 13, 2202–2212 (2007).

    Article  CAS  Google Scholar 

  21. Liphardt, J., Onoa, B., Smith, S.B., Tinoco, I. Jr. & Bustamante, C. Reversible unfolding of single RNA molecules by mechanical force. Science 292, 733–737 (2001).

    Article  CAS  Google Scholar 

  22. Onoa, B. et al. Identifying kinetic barriers to mechanical unfolding of the T. thermophila ribozyme. Science 299, 1892–1895 (2003).

    Article  CAS  Google Scholar 

  23. Noeske, J., Schwalbe, H. & Wöhnert, J. Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain. Nucleic Acids Res. 35, 5262–5273 (2007).

    Article  CAS  Google Scholar 

  24. Allnér, O., Nilsson, L. & Villa, A. Loop-loop interaction in an adenine-sensing riboswitch: a molecular dynamics study. RNA 19, 916–926 (2013).

    Article  Google Scholar 

  25. Stoddard, C.D., Gilbert, S.D. & Batey, R.T. Ligand-dependent folding of the three-way junction in the purine riboswitch. RNA 14, 675–684 (2008).

    Article  CAS  Google Scholar 

  26. Martick, M. & Scott, W.G. Tertiary contacts distant from the active site prime a ribozyme for catalysis. Cell 126, 309–320 (2006).

    Article  CAS  Google Scholar 

  27. Neuman, K.C., Abbondanzieri, E.A., Landick, R., Gelles, J. & Block, S.M. Ubiquitous transcriptional pausing is independent of RNA polymerase backtracking. Cell 115, 437–447 (2003).

    Article  CAS  Google Scholar 

  28. Forde, N.R., Izhaky, D., Woodcock, G.R., Wuite, G.J. & Bustamante, C. Using mechanical force to probe the mechanism of pausing and arrest during continuous elongation by Escherichia coli RNA polymerase. Proc. Natl. Acad. Sci. USA 99, 11682–11687 (2002).

    Article  CAS  Google Scholar 

  29. Tinoco, I. Jr. & Bustamante, C. The effect of force on thermodynamics and kinetics of single molecule reactions. Biophys. Chem. 101-102, 513–533 (2002).

    Article  CAS  Google Scholar 

  30. Bell, G.I. Models for the specific adhesion of cells to cells. Science 200, 618–627 (1978).

    Article  CAS  Google Scholar 

  31. Evans, E. & Ritchie, K. Dynamic strength of molecular adhesion bonds. Biophys. J. 72, 1541–1555 (1997).

    Article  CAS  Google Scholar 

  32. Berg-Sorensen, K. & Flyvjerg, H. Power spectrum analysis for optical tweezers. Rev. Sci. Instrum. 75, 594 (2004).

    Article  CAS  Google Scholar 

  33. Smith, S.B., Cui, Y. & Bustamante, C. Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science 271, 795–799 (1996).

    Article  CAS  Google Scholar 

  34. Wang, M.D., Yin, H., Landick, R., Gelles, J. & Block, S.M. Stretching DNA with optical tweezers. Biophys. J. 72, 1335–1346 (1997).

    Article  CAS  Google Scholar 

  35. Wen, J.D. et al. Force unfolding kinetics of RNA using optical tweezers. I. Effects of experimental variables on measured results. Biophys. J. 92, 2996–3009 (2007).

    Article  CAS  Google Scholar 

  36. Manosas, M. et al. Force unfolding kinetics of RNA using optical tweezers. II. Modeling experiments. Biophys. J. 92, 3010–3021 (2007).

    Article  CAS  Google Scholar 

  37. Žoldák, G., Stigler, J., Pelz, B., Li, H. & Rief, M. Ultrafast folding kinetics and cooperativity of villin headpiece in single-molecule force spectroscopy. Proc. Natl. Acad. Sci. USA 110, 18156–18161 (2013).

    Article  Google Scholar 

  38. Neupane, K. et al. Direct observation of transition paths during the folding of proteins and nucleic acids. Science 352, 239–242 (2016).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank S. Kumar for technical assistance with the RNA preparation for ITC experiments and B. Plaut for writing Matlab codes. We gratefully acknowledge. S. Smith for helping us with the mini-tweezers. We also acknowledge the Protein Data Bank for letting us use the 3D structure of xpt–pbuX guanine riboswitch aptamer (ID: 4FE5) for illustration purpose. This research was generously supported by NSF CAREER (CHE-1151815) awarded to M.M. and support from Single-Molecule and RNA Biology Institute.

Author information

Author notes

  1. Vishnu Chandra, Zain Hannan & Maumita Mandal

    Present address: Present addresses: Rutgers New Jersey Medical School, Newark, New Jersey, USA (V.C.), Department of Chemistry, Swarthmore College, Swarthmore, Pennsylvania, USA (Z.H.) and Single-Molecule and RNA Biology Institute, Pittsburgh, Pennsylvania, USA (M.M.).,

  2. Vishnu Chandra, Zain Hannan and Huizhong Xu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA

    Vishnu Chandra, Zain Hannan & Maumita Mandal

  2. Department of Physics, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA

    Huizhong Xu & Maumita Mandal

  3. Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA

    Maumita Mandal

Authors
  1. Vishnu Chandra
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zain Hannan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Huizhong Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maumita Mandal
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

V.C., H.X. and M.M. collected data in optical tweezers; Z.H. performed the ITC experiments; V.C., H.X., Z.H. and M.M. analyzed data; M.M. designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Maumita Mandal.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–2 and Supplementary Figures 1–6. (PDF 1129 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chandra, V., Hannan, Z., Xu, H. et al. Single-molecule analysis reveals multi-state folding of a guanine riboswitch. Nat Chem Biol 13, 194–201 (2017). https://doi.org/10.1038/nchembio.2252

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2252

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