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Molecular insights into the ligand-controlled organization of the SAM-I riboswitch

Nature Chemical Biology volume 7, pages 384–392 (2011)Cite this article

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Abstract

S-adenosylmethionine (SAM) riboswitches are widespread in bacteria, and up to five different SAM riboswitch families have been reported, highlighting the relevance of SAM regulation. On the basis of crystallographic and biochemical data, it has been postulated, but never demonstrated, that ligand recognition by SAM riboswitches involves key conformational changes in the RNA architecture. We show here that the aptamer follows a two-step hierarchical folding selectively induced by metal ions and ligand binding, each of them leading to the formation of one of the two helical stacks observed in the crystal structure. Moreover, we find that the anti-antiterminator P1 stem is rotated along its helical axis upon ligand binding, a mechanistic feature that could be common to other riboswitches. We also show that the nonconserved P4 helical domain is used as an auxiliary element to enhance the ligand-binding affinity. This work provides the first comprehensive characterization, to our knowledge, of a ligand-controlled riboswitch folding pathway.

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Figure 1: The SAM riboswitch promotes transcription termination upon SAM binding.
Figure 2: The global folding of the SAM riboswitch aptamer studied by CGE and FRET.
Figure 3: Single-molecule FRET analysis of the P1-P3 folding transition.
Figure 4: Mutations inhibiting either pseudoknot formation or SAM binding prevent the folding process in the SAM aptamer.
Figure 5: The P4 stem is important for ligand binding and riboswitch activity.
Figure 6: Helical reorientation of the P1 stem upon SAM binding.
Figure 7: Proposed folding and ligand recognition mechanism of the SAM-I riboswitch.

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References

  1. 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 

  2. Roth, A. & Breaker, R.R. The structural and functional diversity of metabolite-binding riboswitches. Annu. Rev. Biochem. 78, 305–334 (2009).

    Article  CAS  Google Scholar 

  3. Loh, E. et al. A trans-acting riboswitch controls expression of the virulence regulator PrfA in Listeria monocytogenes. Cell 139, 770–779 (2009).

    Article  CAS  Google Scholar 

  4. Schwalbe, H., Buck, J., Furtig, B., Noeske, J. & Wohnert, J. Structures of RNA switches: insight into molecular recognition and tertiary structure. Angew. Chem. Int. Edn Engl. 46, 1212–1219 (2007).

    Article  CAS  Google Scholar 

  5. Edwards, T.E., Klein, D.J. & Ferre-D'Amare, A.R. Riboswitches: small-molecule recognition by gene regulatory RNAs. Curr. Opin. Struct. Biol. 17, 273–279 (2007).

    Article  CAS  Google Scholar 

  6. Dambach, M.D. & Winkler, W.C. Expanding roles for metabolite-sensing regulatory RNAs. Curr. Opin. Microbiol. 12, 161–169 (2009).

    Article  CAS  Google Scholar 

  7. Cromie, M.J., Shi, Y., Latifi, T. & Groisman, E.A. An RNA sensor for intracellular Mg(2+). Cell 125, 71–84 (2006).

    Article  CAS  Google Scholar 

  8. Nechooshtan, G., Elgrably-Weiss, M., Sheaffer, A., Westhof, E. & Altuvia, S. A pH-responsive riboregulator. Genes Dev. 23, 2650–2662 (2009).

    Article  CAS  Google Scholar 

  9. Chowdhury, S., Maris, C., Allain, F.H. & Narberhaus, F. Molecular basis for temperature sensing by an RNA thermometer. EMBO J. 25, 2487–2497 (2006).

    Article  CAS  Google Scholar 

  10. Grundy, F.J. & Henkin, T.M. Regulation of gene expression by effectors that bind to RNA. Curr. Opin. Microbiol. 7, 126–131 (2004).

    Article  CAS  Google Scholar 

  11. Kim, J.N. et al. Design and antimicrobial action of purine analogues that bind Guanine riboswitches. ACS Chem. Biol. 4, 915–927 (2009).

    Article  CAS  Google Scholar 

  12. Mulhbacher, J. et al. Novel riboswitch ligand analogs as selective inhibitors of guanine-related metabolic pathways. PLoS Pathog. 6, e1000865 (2010).

    Article  Google Scholar 

  13. Mulhbacher, J., St-Pierre, P. & Lafontaine, D.A. Therapeutic applications of ribozymes and riboswitches. Curr. Opin. Pharmacol. 10, 551–556 (2010).

    Article  CAS  Google Scholar 

  14. 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).

    Article  CAS  Google Scholar 

  15. 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).

    Article  CAS  Google Scholar 

  16. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. 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).

    Article  CAS  Google Scholar 

  20. 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 

  21. Baird, N.J. & Ferre-D'Amare, A.R. Idiosyncratically tuned switching behavior of riboswitch aptamer domains revealed by comparative small-angle X-ray scattering analysis. RNA 16, 598–609 (2010).

    Article  CAS  Google Scholar 

  22. Epshtein, V., Mironov, A.S. & Nudler, E. The riboswitch-mediated control of sulfur metabolism in bacteria. Proc. Natl. Acad. Sci. USA 100, 5052–5056 (2003).

    Article  CAS  Google Scholar 

  23. 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).

    Article  CAS  Google Scholar 

  24. Lilley, D.M. Analysis of global conformation of branched RNA species using electrophoresis and fluorescence. Methods Enzymol. 317, 368–393 (2000).

    Article  CAS  Google Scholar 

  25. Lumpkin, O.J. Mobility of DNA in gel electrophoresis. Biopolymers 21, 2315–2316 (1982).

    Article  CAS  Google Scholar 

  26. Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nat. Methods 5, 507–516 (2008).

    Article  CAS  Google Scholar 

  27. Weinberg, Z. et al. The aptamer core of SAM-IV riboswitches mimics the ligand-binding site of SAM-I riboswitches. RNA 14, 822–828 (2008).

    Article  CAS  Google Scholar 

  28. Merino, E.J., Wilkinson, K.A., Coughlan, J.L. & Weeks, K.M. RNA structure analysis at single nucleotide resolution by selective 2′-hydroxyl acylation and primer extension (SHAPE). J. Am. Chem. Soc. 127, 4223–4231 (2005).

    Article  CAS  Google Scholar 

  29. 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).

    Article  CAS  Google Scholar 

  30. Garst, A.D., Heroux, A., Rambo, R.P. & Batey, R.T. Crystal structure of the lysine riboswitch regulatory mRNA element. J. Biol. Chem. 283, 22347–22351 (2008).

    Article  CAS  Google Scholar 

  31. 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).

    Article  CAS  Google Scholar 

  32. Noeske, J. et al. Interplay of “induced fit” and preorganization in the ligand induced folding of the aptamer domain of the guanine binding riboswitch. Nucleic Acids Res. 35, 572–583 (2007).

    Article  CAS  Google Scholar 

  33. 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 

  34. Lang, K., Rieder, R. & Micura, R. Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach. Nucleic Acids Res. 35, 5370–5378 (2007).

    Article  CAS  Google Scholar 

  35. 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 

  36. Lipfert, J. et al. Structural transitions and thermodynamics of a glycine-dependent riboswitch from Vibrio cholerae. J. Mol. Biol. 365, 1393–1406 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  39. Gilbert, S.D., Love, C.E., Edwards, A.L. & Batey, R.T. Mutational analysis of the purine riboswitch aptamer domain. Biochemistry 46, 13297–13309 (2007).

    Article  CAS  Google Scholar 

  40. Delfosse, V. et al. Riboswitch structure: an internal residue mimicking the purine ligand. Nucleic Acids Res. 38, 2057–2068 (2010).

    Article  CAS  Google Scholar 

  41. Blouin, S., Chinnappan, R. & Lafontaine, D.A. Folding of the lysine riboswitch: importance of peripheral elements for transcriptional regulation. Nucleic Acids Res., doi:10.1093/nar/gkq1247 (published online 17 December 2010).

    Article  CAS  Google Scholar 

  42. Mahen, E.M., Watson, P.Y., Cottrell, J.W. & Fedor, M.J. mRNA secondary structures fold sequentially but exchange rapidly in vivo. PLoS Biol. 8, e1000307 (2010).

    Article  Google Scholar 

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

    Article  Google Scholar 

  44. Tomsic, J., McDaniel, B.A., Grundy, F.J. & Henkin, T.M. Natural variability in S-adenosylmethionine (SAM)-dependent riboswitches: S-box elements in Bacillus subtilis exhibit differential sensitivity to SAM in vivo and in vitro. J. Bacteriol. 190, 823–833 (2008).

    Article  CAS  Google Scholar 

  45. Wickiser, J.K., Cheah, M.T., Breaker, R.R. & Crothers, D.M. The kinetics of ligand binding by an adenine-sensing riboswitch. Biochemistry 44, 13404–13414 (2005).

    Article  CAS  Google Scholar 

  46. 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).

    Article  CAS  Google Scholar 

  47. Pan, T. & Sosnick, T. RNA folding during transcription. Annu. Rev. Biophys. Biomol. Struct. 35, 161–175 (2006).

    Article  CAS  Google Scholar 

  48. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  Google Scholar 

  49. Brunger, A.T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54, 905–921 (1998).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank A. Lavigueur for critical reading of the manuscript. We also thank D. Norman and P. St.-Pierre for discussion. This work was supported by the Canadian Institutes of Health Research (CIHR). S.B. is supported by a studentship from the National Sciences and Engineering Research Council of Canada. D.A.L. is a CIHR New Investigator scholar as well as a Chercheur-boursier Junior 2 from the Fonds de la recherche en Santé du Québec. J.C.P. thanks the UK Biological and Biotechnological Science Research Council. We are also grateful to T. Ha and S. McKinney (University of Illinois at Urbana-Champaign) for providing the source code software for hidden Markov analysis.

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Authors and Affiliations

  1. Département de biologie, Groupe ARN/RNA Group, Faculté des sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada

    Benoit Heppell, Simon Blouin, Anne-Marie Dussault, Jérôme Mulhbacher & Daniel A Lafontaine

  2. Architecture et réactivité de l'ARN, Université de Strasbourg, Centre national de la recherche scientifique, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France

    Eric Ennifar

  3. School of Physics and Astronomy, University of St. Andrews, St. Andrews, Fife, United Kingdom

    J Carlos Penedo

  4. Biomedical Sciences Research Complex, University of St. Andrews, St. Andrews, Fife, United Kingdom

    J Carlos Penedo

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Contributions

B.H. performed experiments, analyzed data and wrote the paper. S.B. conducted chemical probing. A-M.D. and J.M. carried out comparative gel electrophoresis and in vitro transcription controls, respectively. E.E. performed molecular modeling. J.C.P. and D.A.L. analyzed data and wrote the paper.

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Correspondence to J Carlos Penedo or Daniel A Lafontaine.

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Heppell, B., Blouin, S., Dussault, AM. et al. Molecular insights into the ligand-controlled organization of the SAM-I riboswitch. Nat Chem Biol 7, 384–392 (2011). https://doi.org/10.1038/nchembio.563

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