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:

c-di-AMP binds the ydaO riboswitch in two pseudo-symmetry–related pockets

Nature Chemical Biology volume 10, pages 780–786 (2014)Cite this article

Subjects

Abstract

The ydaO riboswitch, involved in sporulation, osmotic stress responses and cell wall metabolism, targets the second messenger cyclic-di-AMP with subnanomolar affinity. We have solved the structure of c-di-AMP bound to the Thermoanaerobacter tengcongensis ydaO riboswitch, thereby identifying a five-helical scaffold containing a zippered-up bubble, a pseudoknot and long-range tertiary base pairs. Highlights include the identification of two c-di-AMP binding pockets on the same face of the riboswitch, related by pseudo-two-fold symmetry, with potential for cross-talk between sites mediated by adjacently positioned base-stacking alignments connecting pockets. The adenine rings of bound c-di-AMP molecules are wedged between bases and stabilized by stacking, base-sugar and sugar-sugar intermolecular hydrogen bonding interactions. The structural studies are complemented by isothermal titration calorimetry–based binding studies of mutants mediating key tertiary intermolecular contacts. The T. tengcongensis ydaO riboswitch, like its Bacillus subtilis counterpart, most likely functions through a transcription termination mechanism, with the c-di-AMP bound state representing an 'off' switch.

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: Secondary and tertiary structure of the T. tengcongensis ydaO riboswitch bound to c-di-AMP and alignment of c-di-AMP molecules A and B in their respective binding pockets.
Figure 2: Folding topologies adopted by the bubble and pseudoknot segments and their flanking stem segments in the structure of T. tengcongensis ydaO riboswitch bound to c-di-AMP.
Figure 3: Similarities in recognition features between Aα rings of c-di-AMP molecules A and B bound within their respective binding pockets and a pair of parallel base stacking alignments connecting binding pockets in the structure of T. tengcongensis ydaO riboswitch bound to c-di-AMP.
Figure 4: Comparison of ITC binding curves with other second messengers and analogs and mutants involved in tertiary pairing in the T. tengcongensis ydaO riboswitch.
Figure 5: Comparison of binding pockets and intermolecular hydrogen bonding contacts in complexes of c-di-AMP bound to the ydaO riboswitch and the protein DisA.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

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  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Nudler, E. & Miranov, A.S. The riboswitch control of bacterial metabolism. Trends Biochem. Sci. 29, 11–17 (2004).

    Article  CAS  Google Scholar 

  4. Winkler, W.C. & Breaker, R.R. Regulation of bacterial gene expression by riboswitches. Annu. Rev. Microbiol. 59, 487–517 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Serganov, A. & Patel, D.J. Ribozymes, riboswitches and beyond: regulation of gene expression without proteins. Nat. Rev. Genet. 8, 776–790 (2007).

    Article  CAS  Google Scholar 

  8. Serganov, A. & Patel, D.J. Metabolic recognition principles and molecular mechanisms underlying riboswitch function. Annu. Rev. Biophys 41, 343–370 (2012).

    Article  CAS  Google Scholar 

  9. Sudarsan, N. et al. Riboswitches in eubacteria sense the second messenger c-di-GMP. Science 321, 411–413 (2008).

    Article  CAS  Google Scholar 

  10. Lee, E.R. et al. An allosteric self-splicing ribozyme triggered by a bacterial second messenger. Science 329, 845–848 (2010).

    Article  CAS  Google Scholar 

  11. Tamayo, R., Pratt, J.T. & Camilli, A. Roles of cyclic diguanylate in the regulation of bacterial pathogenesis. Annu. Rev. Microbiol. 61, 131–148 (2007).

    Article  CAS  Google Scholar 

  12. Cotter, P.A. & Stibitz, S. c-di-GMP–mediated regulation of virulence and biofilm formation. Curr. Opin. Microbiol. 10, 17–23 (2007).

    Article  CAS  Google Scholar 

  13. Smith, K.D. et al. Structural basis of ligand binding by a c-di-GMP riboswitch. Nat. Struct. Mol. Biol. 16, 1218–1223 (2009).

    Article  CAS  Google Scholar 

  14. Kulshina, N., Baird, N.J. & Ferre-D'Amare, A.R. Recognition of the bacterial second messenger cyclic diguanylate by its cognate riboswitch. Nat. Struct. Mol. Biol. 16, 1212–1217 (2009).

    Article  CAS  Google Scholar 

  15. Smith, K.D. et al. Structural basis of differential ligand recognition by two classes of bis-(3′,5′)-cyclic dimeric guanosine monophosphate-binding riboswitches. Proc. Natl. Acad. Sci. USA 108, 7757–7762 (2011).

    Article  CAS  Google Scholar 

  16. Smith, K.D. & Strobel, S.A. Interactions of the c-di-GMP riboswitch with its second messenger ligand. Biochem. Soc. Trans. 39, 647–651 (2011).

    Article  CAS  Google Scholar 

  17. Barrick, J.E. et al. New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control. Proc. Natl. Acad. Sci. USA 101, 6421–6426 (2004).

    Article  CAS  Google Scholar 

  18. Block, K.F., Hammond, M.C. & Breaker, R.R. Evidence for widespread gene control function by the ydaO riboswitch candidate. J. Bacteriol. 192, 3983–3989 (2010).

    Article  CAS  Google Scholar 

  19. Watson, P.Y. & Fedor, M.J. The ydaO motif is an ATP-sensing riboswitch in Bacillus subtilis. Nat. Chem. Biol. 8, 963–965 (2012).

    Article  CAS  Google Scholar 

  20. Nelson, J.W. et al. Riboswitches in eubacteria sense the second messenger c-di-AMP. Nat. Chem. Biol. 9, 834–839 (2013).

    Article  CAS  Google Scholar 

  21. Witte, G., Hartung, S., Büttner, K. & Hopfner, K.P. Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates. Science 329, 845–848 (2010).

    Article  Google Scholar 

  22. Corrigan, R.M. et al. Systematic identification of conserved bacterial c-di-AMP receptor proteins. Proc. Natl. Acad. Sci. USA 110, 9084–9089 (2013).

    Article  CAS  Google Scholar 

  23. Corrigan, R.M. et al. c-di-AMP is a new second messenger in S. aureus with a role in controlling cell size and envelop stress. PLoS Pathog. 7, e1002217 (2011).

    Article  CAS  Google Scholar 

  24. Fong, J.C. et al. Interplay between cyclic AMP-cyclic AMP receptor protein and cyclic di-GMP signaling in Vibrio cholera biofilm formation. J. Bacteriol. 190, 6646–6659 (2008).

    Article  CAS  Google Scholar 

  25. Gao, P. et al. Cyclic [G(2′,5′)pA(3′,5′)p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase. Cell 153, 1094–1107 (2013).

    Article  CAS  Google Scholar 

  26. Diner, E.J. et al. The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING. Cell Reports 3, 1355–1361 (2013).

    Article  CAS  Google Scholar 

  27. Ablasser, A. et al. cGAS produces a 2′,5′-linked cyclic dinucleotide second messenger that activates STING. Nature 498, 380–384 (2013).

    Article  CAS  Google Scholar 

  28. Zhang, X. et al. Cyclic GMP-AMP containing mixed phosphodiester linkage is an endogenous high-affinity ligand for STING. Mol. Cell 51, 226–235 (2013).

    Article  CAS  Google Scholar 

  29. Serganov, A., Huang, L. & Patel, D.J. Coenzyme recognition and gene regulation by a FMN riboswitch. Nature 458, 233–237 (2009).

    Article  CAS  Google Scholar 

  30. Huang, L., Ishibe-Murakami, S., Patel, D.J. & Serganov, A. Long-range pseudoknot interactions dictate the regulatory response in the tetrahydrofolate riboswitch. Proc. Natl. Acad. Sci. USA 108, 14801–14806 (2011).

    Article  CAS  Google Scholar 

  31. Trausch, J.J., Ceres, P., Reyes, F.E. & Batey, R.T. The structure of a tetrahydrofolate-sensing riboswitch reveals two ligand-binding sites in a single aptamer. Structure 19, 1413–1423 (2011).

    Article  CAS  Google Scholar 

  32. Pikovskaya, O. et al. Preparation and crystallization of riboswitch-ligand complexes. Methods Mol. Biol. 540, 115–128 (2009).

    Article  CAS  Google Scholar 

  33. Pape, T. & Schneider, T.R. HKL2MAP: a graphical user interface for phasing with SHELX programs. J. Appl. Crystallogr. 37, 843–844 (2004).

    Article  CAS  Google Scholar 

  34. Adams, P.D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002).

    Article  Google Scholar 

  35. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  36. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge assistance by staff at the X-29 beamline at the Brookhaven National Laboratory and NE-CAT beamlines at the Advanced Photon Source. We thank B. Gaffney and R. Jones of Rutgers University for samples of chemically synthesized cGAMP linkage isomers. The research was supported by US National Institutes of Health grant 1 U19 CA179564 to D.J.P.

Author information

Authors and Affiliations

  1. Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Aiming Ren & Dinshaw J Patel

Authors
  1. Aiming Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dinshaw J Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.R. undertook all of the crystallographic and ITC experiments under the supervision of D.J.P.

Corresponding author

Correspondence to Dinshaw J Patel.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–19. (PDF 38749 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ren, A., Patel, D. c-di-AMP binds the ydaO riboswitch in two pseudo-symmetry–related pockets. Nat Chem Biol 10, 780–786 (2014). https://doi.org/10.1038/nchembio.1606

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.1606

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