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:

An excited state underlies gene regulation of a transcriptional riboswitch

Nature Chemical Biology volume 13, pages 968–974 (2017)Cite this article

Subjects

Abstract

Riboswitches control gene expression through ligand-dependent structural rearrangements of the sensing aptamer domain. However, we found that the Bacillus cereus fluoride riboswitch aptamer adopts identical tertiary structures in solution with and without ligand. Using chemical-exchange saturation transfer (CEST) NMR spectroscopy, we revealed that the structured ligand-free aptamer transiently accesses a low-populated (∼1%) and short-lived (∼3 ms) excited conformational state that unravels a conserved 'linchpin' base pair to signal transcription termination. Upon fluoride binding, this highly localized, fleeting process is allosterically suppressed, which activates transcription. We demonstrated that this mechanism confers effective fluoride-dependent gene activation over a wide range of transcription rates, which is essential for robust toxicity responses across diverse cellular conditions. These results unveil a novel switching mechanism that employs ligand-dependent suppression of an aptamer excited state to coordinate regulatory conformational transitions rather than adopting distinct aptamer ground-state tertiary architectures, exemplifying a new mode of ligand-dependent RNA regulation.

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: Solution structure of the apo B. cereus fluoride riboswitch aptamer.
Figure 2: The fluoride riboswitch aptamer adopts structurally similar apo and holo states.
Figure 3: The apo B. cereus fluoride riboswitch aptamer populates an excited state.
Figure 4: Single-round transcription assay of the B. cereus fluoride riboswitch in the absence of fluoride.
Figure 5: Transcription regulation by the B. cereus fluoride riboswitch.

Similar content being viewed by others

Accession codes

Primary accessions

Biological Magnetic Resonance Data Bank

Protein Data Bank

References

  1. Mironov, A.S. et al. Sensing small molecules by nascent RNA: a mechanism to control transcription in bacteria. Cell 111, 747–756 (2002).

    Article  CAS  PubMed  Google Scholar 

  2. Nahvi, A. et al. Genetic control by a metabolite binding mRNA. Chem. Biol. 9, 1043 (2002).

    CAS  PubMed  Google Scholar 

  3. Winkler, W., Nahvi, A. & Breaker, R.R. Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419, 952–956 (2002).

    CAS  PubMed  Google Scholar 

  4. Serganov, A. & Nudler, E. A decade of riboswitches. Cell 152, 17–24 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 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 

  9. Haller, A., Rieder, U., Aigner, M., Blanchard, S.C. & Micura, R. Conformational capture of the SAM-II riboswitch. Nat. Chem. Biol. 7, 393–400 (2011).

    CAS  PubMed  Google Scholar 

  10. Wilson, R.C. et al. Tuning riboswitch regulation through conformational selection. J. Mol. Biol. 405, 926–938 (2011).

    CAS  PubMed  Google Scholar 

  11. 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 

  12. Chen, B., Zuo, X., Wang, Y.X. & Dayie, T.K. Multiple conformations of SAM-II riboswitch detected with SAXS and NMR spectroscopy. Nucleic Acids Res. 40, 3117–3130 (2012).

    CAS  PubMed  Google Scholar 

  13. Suddala, K.C. et al. Single transcriptional and translational preQ1 riboswitches adopt similar pre-folded ensembles that follow distinct folding pathways into the same ligand-bound structure. Nucleic Acids Res. 41, 10462–10475 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  15. Zhang, J., Jones, C.P. & Ferré-D'Amaré, A.R. Global analysis of riboswitches by small-angle X-ray scattering and calorimetry. Biochim. Biophys. Acta 1839, 1020–1029 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Ren, A. et al. Structural and dynamic basis for low-affinity, high-selectivity binding of L-glutamine by the glutamine riboswitch. Cell Rep. 13, 1800–1813 (2015).

    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. Serganov, A., Huang, L. & Patel, D.J. Structural insights into amino acid binding and gene control by a lysine riboswitch. Nature 455, 1263–1267 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Huang, L., Serganov, A. & Patel, D.J. Structural insights into ligand recognition by a sensing domain of the cooperative glycine riboswitch. Mol. Cell 40, 774–786 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Jenkins, J.L., Krucinska, J., McCarty, R.M., Bandarian, V. & Wedekind, J.E. Comparison of a preQ1 riboswitch aptamer in metabolite-bound and free states with implications for gene regulation. J. Biol. Chem. 286, 24626–24637 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Vicens, Q., Mondragón, E. & Batey, R.T. Molecular sensing by the aptamer domain of the FMN riboswitch: a general model for ligand binding by conformational selection. Nucleic Acids Res. 39, 8586–8598 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Stagno, J.R. et al. Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography. Nature 541, 242–246 (2017).

    CAS  PubMed  Google Scholar 

  23. Baker, J.L. et al. Widespread genetic switches and toxicity resistance proteins for fluoride. Science 335, 233–235 (2012).

    CAS  PubMed  Google Scholar 

  24. Li, S. et al. Eukaryotic resistance to fluoride toxicity mediated by a widespread family of fluoride export proteins. Proc. Natl. Acad. Sci. USA 110, 19018–19023 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Ren, A., Rajashankar, K.R. & Patel, D.J. Fluoride ion encapsulation by Mg2+ ions and phosphates in a fluoride riboswitch. Nature 486, 85–89 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Bothe, J.R. et al. Characterizing RNA dynamics at atomic resolution using solution-state NMR spectroscopy. Nat. Methods 8, 919–931 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 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 

  28. Sekhar, A. & Kay, L.E. NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers. Proc. Natl. Acad. Sci. USA 110, 12867–12874 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Palmer, A.G. III. Chemical exchange in biomacromolecules: past, present, and future. J. Magn. Reson. 241, 3–17 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Dethoff, E.A., Petzold, K., Chugh, J., Casiano-Negroni, A. & Al-Hashimi, H.M. Visualizing transient low-populated structures of RNA. Nature 491, 724–728 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Fawzi, N.L., Ying, J., Ghirlando, R., Torchia, D.A. & Clore, G.M. Atomic-resolution dynamics on the surface of amyloid-β protofibrils probed by solution NMR. Nature 480, 268–272 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Vallurupalli, P., Bouvignies, G. & Kay, L.E. Studying “invisible” excited protein states in slow exchange with a major state conformation. J. Am. Chem. Soc. 134, 8148–8161 (2012).

    CAS  PubMed  Google Scholar 

  33. Zhao, B., Hansen, A.L. & Zhang, Q. Characterizing slow chemical exchange in nucleic acids by carbon CEST and low spin-lock field R(1ρ) NMR spectroscopy. J. Am. Chem. Soc. 136, 20–23 (2014).

    CAS  PubMed  Google Scholar 

  34. Monforte, J.A., Kahn, J.D. & Hearst, J.E. RNA folding during transcription by Escherichia coli RNA polymerase analyzed by RNA self-cleavage. Biochemistry 29, 7882–7890 (1990).

    CAS  PubMed  Google Scholar 

  35. Komissarova, N. & Kashlev, M. Functional topography of nascent RNA in elongation intermediates of RNA polymerase. Proc. Natl. Acad. Sci. USA 95, 14699–14704 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Gusarov, I. & Nudler, E. Control of intrinsic transcription termination by N and NusA: the basic mechanisms. Cell 107, 437–449 (2001).

    CAS  PubMed  Google Scholar 

  37. Yarnell, W.S. & Roberts, J.W. Mechanism of intrinsic transcription termination and antitermination. Science 284, 611–615 (1999).

    CAS  PubMed  Google Scholar 

  38. Gusarov, I. & Nudler, E. The mechanism of intrinsic transcription termination. Mol. Cell 3, 495–504 (1999).

    CAS  PubMed  Google Scholar 

  39. Abbondanzieri, E.A., Greenleaf, W.J., Shaevitz, J.W., Landick, R. & Block, S.M. Direct observation of base-pair stepping by RNA polymerase. Nature 438, 460–465 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Watters, K.E., Strobel, E.J., Yu, A.M., Lis, J.T. & Lucks, J.B. Cotranscriptional folding of a riboswitch at nucleotide resolution. Nat. Struct. Mol. Biol. 23, 1124–1131 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Wang, J.X., Lee, E.R., Morales, D.R., Lim, J. & Breaker, R.R. Riboswitches that sense S-adenosylhomocysteine and activate genes involved in coenzyme recycling. Mol. Cell 29, 691–702 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Nelson, J.W., Atilho, R.M., Sherlock, M.E., Stockbridge, R.B. & Breaker, R.R. Metabolism of free guanidine in bacteria is regulated by a widespread riboswitch class. Mol. Cell 65, 220–230 (2017).

    CAS  PubMed  Google Scholar 

  43. Tzeng, S.R. & Kalodimos, C.G. Allosteric inhibition through suppression of transient conformational states. Nat. Chem. Biol. 9, 462–465 (2013).

    CAS  PubMed  Google Scholar 

  44. Dethoff, E.A., Chugh, J., Mustoe, A.M. & Al-Hashimi, H.M. Functional complexity and regulation through RNA dynamics. Nature 482, 322–330 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Hoogstraten, C.G., Wank, J.R. & Pardi, A. Active site dynamics in the lead-dependent ribozyme. Biochemistry 39, 9951–9958 (2000).

    CAS  PubMed  Google Scholar 

  46. Blad, H., Reiter, N.J., Abildgaard, F., Markley, J.L. & Butcher, S.E. Dynamics and metal ion binding in the U6 RNA intramolecular stem-loop as analyzed by NMR. J. Mol. Biol. 353, 540–555 (2005).

    CAS  PubMed  Google Scholar 

  47. Henzler-Wildman, K. & Kern, D. Dynamic personalities of proteins. Nature 450, 964–972 (2007).

    CAS  PubMed  Google Scholar 

  48. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    CAS  PubMed  Google Scholar 

  49. Zhang, Q., Kim, N.K., Peterson, R.D., Wang, Z. & Feigon, J. Structurally conserved five nucleotide bulge determines the overall topology of the core domain of human telomerase RNA. Proc. Natl. Acad. Sci. USA 107, 18761–18768 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Duchardt-Ferner, E., Ferner, J. & Wöhnert, J. Rapid identification of noncanonical RNA structure elements by direct detection of OH⋯O=P, NH⋯O=P, and NH2⋯O=P hydrogen bonds in solution NMR spectroscopy. Angew. Chem. Int. Edn. Engl. 50, 7927–7930 (2011).

    CAS  Google Scholar 

  51. Bermejo, G.A., Clore, G.M. & Schwieters, C.D. Improving NMR Structures of RNA. Structure 24, 806–815 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Koradi, R., Billeter, M. & Wüthrich, K. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55, 29–32 (1996).

    CAS  PubMed  Google Scholar 

  53. Davis, I.W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).

    PubMed  PubMed Central  Google Scholar 

  54. Johnson, B.A. & Blevins, R.A. NMR View: a computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994).

    CAS  PubMed  Google Scholar 

  55. McConnell, H.M. Reaction rates by nuclear magnetic resonance. J. Chem. Phys. 28, 430–431 (1958).

    CAS  Google Scholar 

  56. Vallurupalli, P. & Kay, L.E. Probing slow chemical exchange at carbonyl sites in proteins by chemical exchange saturation transfer NMR spectroscopy. Angew. Chem. Int. Edn. Engl. 52, 4156–4159 (2013).

    CAS  Google Scholar 

  57. Landick, R., Wang, D. & Chan, C.L. Quantitative analysis of transcriptional pausing by Escherichia coli RNA polymerase: his leader pause site as paradigm. Methods Enzymol. 274, 334–353 (1996).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Mejia, Y.X., Nudler, E. & Bustamante, C. Trigger loop folding determines transcription rate of Escherichia coli's RNA polymerase. Proc. Natl. Acad. Sci. USA 112, 743–748 (2015).

    CAS  PubMed  Google Scholar 

  60. Xu, X., Yu, T. & Chen, S.J. Understanding the kinetic mechanism of RNA single base pair formation. Proc. Natl. Acad. Sci. USA 113, 116–121 (2016).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank G. Young for maintenance of NMR instruments and members of the Zhang lab for critical comments. This work was supported by start-up fund from the University of North Carolina at Chapel Hill and an NIH grant (R01 GM114432) to Q.Z.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Bo Zhao, Sharon L Guffy, Benfeard Williams & Qi Zhang

  2. Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Bo Zhao

Authors
  1. Bo Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sharon L Guffy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Benfeard Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qi Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

B.Z. and Q.Z. conceived the project and experimental design. B.Z. and Q.Z. prepared the samples, carried out NMR and biochemical experiments, analyzed the data, and wrote the paper. S.L.G., B.W., and Q.Z. analyzed NMR RDC data.

Corresponding author

Correspondence to Qi Zhang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–12 and Supplementary Table 1 (PDF 7656 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhao, B., Guffy, S., Williams, B. et al. An excited state underlies gene regulation of a transcriptional riboswitch. Nat Chem Biol 13, 968–974 (2017). https://doi.org/10.1038/nchembio.2427

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2427

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