Conformational capture of the SAM-II riboswitch

Journal name:
Nature Chemical Biology
Volume:
7,
Pages:
393–400
Year published:
DOI:
doi:10.1038/nchembio.562
Received
Accepted
Published online

Abstract

Riboswitches are gene regulation elements in mRNA that function by specifically responding to metabolites. Although the metabolite-bound states of riboswitches have proven amenable to structure determination efforts, knowledge of the structural features of riboswitches in their ligand-free forms and their ligand-response mechanisms giving rise to regulatory control is lacking. Here we explore the ligand-induced folding process of the S-adenosylmethionine type II (SAM-II) riboswitch using chemical and biophysical methods, including NMR and fluorescence spectroscopy, and single-molecule fluorescence imaging. The data reveal that the unliganded SAM-II riboswitch is dynamic in nature, in that its stem-loop element becomes engaged in a pseudoknot fold through base-pairing with nucleosides in the 3′ overhang containing the Shine-Dalgarno sequence. Although the pseudoknot structure is highly transient in the absence of its ligand, S-adenosylmethionine (SAM), it becomes conformationally restrained upon ligand recognition, through a conformational capture mechanism. These insights provide a molecular understanding of riboswitch dynamics that shed new light on the mechanism of riboswitch-mediated translational regulation.

At a glance

Figures

  1. Class-II SAM riboswitch.
    Figure 1: Class-II SAM riboswitch.

    (a) Cartoon representation of the global structure of the metX SAM-II RNA bound to SAM8 (PDB ID: 2QWY). (b) Secondary-tertiary structure interactions in Leontis-Westhof nomenclature50. (c) Minimal secondary structure model of the unliganded SAM-II RNA. Shine-Dalgarno sequence, SD.

  2. Pseudoknot interaction analyzed by labeled SAM-II RNA using NMR and fluorescence spectroscopy.
    Figure 2: Pseudoknot interaction analyzed by labeled SAM-II RNA using NMR and fluorescence spectroscopy.

    (a) Schematics of SAM-II pseudoknot with crucial base pair C16-G50. (b) 1H-NMR imino proton spectra of unlabeled SAM-II RNA in the absence of ligand and Mg2+ (top), after addition of Mg2+ (middle) and after addition of SAM (bottom). (c) 15N-labeled cytidine-16 SAM-II variant. (d) First increments of 1H/15N HSQC spectra of C16-15N(4) RNA without (top) and with (middle) SAM (the reference spectrum of RNA in buffer (no Mg2+, no SAM) was identical to the one with RNA and Mg2+). 1H/15N HSQC spectrum of C16-15N(4) RNA in the presence of SAM (two-dimensional plot, bottom). Asterisk indicates residual signal of ammonium buffer. (e) 19F-labeled cytidine-16 SAM-II variant. (f) 19F NMR spectra of 5-fluoro-C16 labeled RNA in absence of ligand and absence of Mg2+ (top), after addition of Mg2+ (middle), and after addition of SAM (bottom); for 1H-NMR imino proton spectra of 5-fluoro-C16 labeled RNA see Supplementary Figure 7. NMR conditions: cRNA = 0.2 mM, cSAM = 0.4 mM, cMg = 2.0 mM, 25 mM Na2HAsO4, pH 7.0, H2O/D2O 9:1, 298 K for b and f and 288 K for d. (g) Schematics of Cy3-Cy5–labeled SAM-II variant (upper panel) and fluorescence emission response of acceptor dye upon Mg2+ and SAM addition. See Supplementary Figure 2 for structures of fluorophores and linkers. Fluorescence spectroscopy conditions: cRNA = 0.5 μM, cSAM = 5.0 μM, cMg = 2.0 mM, 50 mM KMOPS, 100 mM KCl, 20 °C, pH 7.5; λex = 540 nm, λem = 665 nm.

  3. Dynamics of pseudoknot formation of the SAM-II riboswitch analyzed by smFRET experiments.
    Figure 3: Dynamics of pseudoknot formation of the SAM-II riboswitch analyzed by smFRET experiments.

    (a) Upper panel: population FRET histograms showing the mean FRET values and percent (%) occupancies of each state observed for the SAM-II riboswitch in the absence of Mg2+ and SAM ligand. Lower panels: Fluorescence (green-Cy3; red-Cy5) and FRET (blue) trajectories of individual SAM-II riboswitch molecules under the same conditions, where idealization of the data to a two-state Markov chain is shown in red. (b) Same as a but in the presence of 2 mM Mg2+ ions. (c) Same as a but in the presence of 2 mM Mg2+ ions and 10 μM SAM.

  4. SAM-II variants containing single 2-aminopurine labels: structural analysis and fluorescence response.
    Figure 4: SAM-II variants containing single 2-aminopurine labels: structural analysis and fluorescence response.

    (a) Replacements of U34: position within the aptamer (left), local chemical environment (middle) and fluorescence response of the U34AP variant upon Mg2+ (2 mM) and subsequent SAM (5 μM) addition (right); start conditions: cRNA = 0.5 μM, 50 mM KMOPS, 100 mM KCl, 20 °C, pH 7.5; both additions (Mg2+ and SAM) were performed manually (with an operational time of ∼4 s). (b) Replacement of A41. (c) Replacement of A14. Conditions: cRNA = 0.5 μM, cSAM = 5.0 μM, cMg = 2.0 mM, 50 mM KMOPS, 100 mM KCl, 20 °C, pH 7.5; λex = 308 nm, λem = 372 nm.

  5. Thermodynamics and kinetics of the ligand-induced SAM-II riboswitch response.
    Figure 5: Thermodynamics and kinetics of the ligand-induced SAM-II riboswitch response.

    (a) Fluorescence changes upon titration of the U34Ap-labeled variant with SAM. Normalized Ap fluorescence intensity plotted as a function of SAM concentration. The graph shows the best fit to a single-site binding model (see Supplementary Methods). The inset shows fluorescence emission spectra (λex = 308 nm) from 320 to 480 nm for each SAM concentration. (b) Fluorescence changes upon titration of the three individually Ap labeled SAM-II variants with Mg2+ in the absence of SAM. Normalized Ap fluorescence intensity plotted as a function of Mg2+ concentration (logarithmic scale). (c) Time course of fluorescence response exemplified for the U34Ap variant. Conditions: cRNA = 0.3 μM, cSAM = 3.0 μM, 50 mM KMOPS, 100 mM KCl, 2 mM MgCl2, 20 °C, pH 7.5; mixing was performed with a stopped-flow apparatus. (d) Graphical representation of the different magnitudes of rate constants k for individual Ap and the Cy5-Cy3 variants and their correlation to the label position and the corresponding secondary structure element (data represent mean values ± s.d.).

  6. Folding model of the SAM-II riboswitch.
    Figure 6: Folding model of the SAM-II riboswitch.

    The experimental data support a model where Mg2+ ions stabilize the P1/L3 segment of the riboswitch to a significant extent, thereby also supporting the preorganization of loop L1 together with the binding pocket (II). Moreover, in the presence of Mg2+, the transient pseudoknot-like folds in the ensemble III reach ten-fold increased lifetimes (subsecond region). SAM selects (or 'captures') fold III and adaptive, conformational rearrangements occur in distinct order and even in distal regions to the binding site. The culmination of the process is formation of stem P2a which masks the functionally crucial Shine-Dalgarno (SD) sequence (IV) and thereby renders recognition by the ribosome unfeasible, hampering translation initiation.

Accession codes

Referenced accessions

Protein Data Bank

References

  1. Roth, A. & Breaker, R.R. The structural and functional diversity of metabolite-binding riboswitches. Annu. Rev. Biochem. 78, 305334 (2009).
  2. Montange, R.K. & Batey, R.T. Riboswitches: emerging themes in RNA structure and function. Annu. Rev. Biophys 37, 117133 (2008).
  3. Blouin, S., Mulhbacher, J., Penedo, J.C. & Lafontaine, D.A. Riboswitches: ancient and promising genetic regulators. ChemBioChem 10, 400416 (2009).
  4. Serganov, A. & Patel, D.J. Ribozymes, riboswitches and beyond: regulation of gene expression without proteins. Nat. Rev. Genet. 8, 776790 (2007).
  5. Schwalbe, H., Buck, J., Fürtig, B., Noeske, J. & Wöhnert, J. Structures of RNA switches: insight into molecular recognition and tertiary structure. Angew. Chem. Int. Edn. Engl. 46, 12121219 (2007).
  6. Nudler, E. & Mironov, A.S. The riboswitch control of bacterial metabolism. Trends Biochem. Sci. 29, 1117 (2004).
  7. Serganov, A. Determination of riboswitch structures: light at the end of the tunnel? RNA Biol. 7, 98103 (2010).
  8. Gilbert, S.D., Rambo, R.P., Van Tyne, D. & Batey, R.T. Structure of the SAM-II riboswitch bound to S-adenosylmethionine. Nat. Struct. Mol. Biol. 15, 177182 (2008).
  9. Garst, A.D. & Batey, R.T. A switch in time: detailing the life of a riboswitch. Biochim. Biophys. Acta 1789, 584591 (2009).
  10. Stoddard, C.D. et al. Free state conformational sampling of the SAM-I riboswitch aptamer domain. Structure 18, 787797 (2010).
  11. Baird, N.J. & Ferré-D'Amaré, A.R. Idiosyncratically tuned switching behavior of riboswitch aptamer domains revealed by comparative small-angle X-ray scattering analysis. RNA 16, 598609 (2010).
  12. Edwards, A.L., Reyes, F.E., Héroux, A. & Batey, R.T. Structural basis for recognition of S-adenosylhomocysteine by riboswitches. RNA 16, 21442155 (2010).
  13. Bosshard, H.R. Molecular recognition by induced fit: how fit is the concept? News Physiol. Sci. 16, 171173 (2001).
  14. Weikl, T.R. & von Deuster, C. Selected-fit versus induced-fit protein binding: Kinetic differences and mutational analysis. Proteins 75, 104110 (2009).
  15. 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, 572583 (2007).
  16. Boehr, D.D., Nussinov, R. & Wright, P.E. The role of dynamic conformational ensembles in biomolecular recognition. Nat. Chem. Biol. 5, 789796 (2009).
  17. Leulliot, N. & Varani, G. Current topics in RNA-protein recognition: control of specificity and biological function through induced fit and conformational capture. Biochemistry 40, 79477956 (2001).
  18. Duchardt-Ferner, E. et al. Highly modular structure and ligand binding by conformational capture in a minimalistic riboswitch. Angew. Chem. Int. Edn Engl. 49, 62166219 (2010).
  19. Stelzer, A.C., Kratz, J.D., Zhang, Q. & Al-Hashimi, H.M. RNA dynamics by design: biasing ensembles towards the ligand-bound state. Angew. Chem. Int. Edn Engl. 49, 57315733 (2010).
  20. Wang, J.X. & Breaker, R.R. Riboswitches that sense S-adenosylmethionine and S-adenosylhomocysteine. Biochem. Cell Biol. 86, 157168 (2008).
  21. Poiata, E., Meyer, M.M., Ames, T.D. & Breaker, R.R. A variant riboswitch aptamer class for S-adenosylmethionine common in marine bacteria. RNA 15, 20462056 (2009).
  22. Corbino, K.A. et al. Evidence for a second class of S-adenosylmethionine riboswitches and other regulatory RNA motifs in alpha-proteobacteria. Genome Biol. 6, R70 (2005).
  23. Rieder, U., Lang, K., Kreutz, C., Polacek, N. & Micura, R. Evidence for pseudoknot formation of class I preQ1 riboswitch aptamers. ChemBioChem 10, 11411144 (2009).
  24. Puffer, B. et al. 5-Fluoro pyrimidines: labels to probe DNA and RNA secondary structures by 1D 19F NMR spectroscopy. Nucleic Acids Res. 37, 77287740 (2009).
  25. Rieder, U., Kreutz, C. & Micura, R. Folding of a transcriptionally acting preQ1 riboswitch. Proc. Natl. Acad. Sci. USA 107, 1080410809 (2010).
  26. Lang, K. & Micura, R. The preparation of site-specifically modified riboswitch domains as an example for enzymatic ligation of chemically synthesized RNA fragments. Nat. Protoc. 3, 14571466 (2008).
  27. Alemán, E.A., Lamichhane, R. & Rueda, D. Exploring RNA folding one molecule at a time. Curr. Opin. Chem. Biol. 12, 647654 (2008).
  28. Lemay, J.F., Penedo, J.C., Tremblay, R., Lilley, D.M. & Lafontaine, D.A. Folding of the adenine riboswitch. Chem. Biol. 13, 857868 (2006).
  29. 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, 15961605 (2010).
  30. Munro, J.B., Altman, R.B., O'Connor, N. & Blanchard, S.C. Identification of two distinct hybrid state intermediates on the ribosome. Mol. Cell 25, 505517 (2007).
  31. 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, 53705378 (2007).
  32. 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, 896902 (2007).
  33. Kelley, J.M. & Hamelberg, D. Atomistic basis for the on-off signaling mechanism in SAM-II riboswitch. Nucleic Acids Res. 38, 13921400 (2010).
  34. Mortimer, S.A. & Weeks, K.M. A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry. J. Am. Chem. Soc. 129, 41444145 (2007).
  35. Weeks, K.M. Advances in RNA structure analysis by chemical probing. Curr. Opin. Struct. Biol. 20, 295304 (2010).
  36. Staple, D.W. & Butcher, S.E. Pseudoknots: RNA structures with diverse functions. PLoS Biol. 3, e213 (2005).
  37. Serganov, A., Ennifar, E., Portier, C., Ehresmann, B. & Ehresmann, C. Do mRNA and rRNA binding sites of E.coli ribosomal protein S15 share common structural determinants? J. Mol. Biol. 320, 963978 (2002).
  38. Munro, J.B., Wasserman, M.R., Altman, R.B., Wang, L. & Blanchard, S.C. Correlated conformational events in EF-G and the ribosome regulate translocation. Nat. Struct. Mol. Biol. 17, 14701477 (2010).
  39. Al-Hashimi, H.M. & Walter, N.G. RNA dynamics: it is about time. Curr. Opin. Struct. Biol. 18, 321329 (2008).
  40. Zhang, Q., Stelzer, A.C., Fisher, C.K. & Al-Hashimi, H.M. Visualizing spatially correlated dynamics that directs RNA conformational transitions. Nature 450, 12631267 (2007).
  41. Frank, A.T., Stelzer, A.C., Al-Hashimi, H.M. & Andricioaei, I. Constructing RNA dynamical ensembles by combining MD and motionally decoupled NMR RDCs: new insights into RNA dynamics and adaptive ligand recognition. Nucleic Acids Res. 37, 36703679 (2009).
  42. Lu, C. et al. SAM recognition and conformational switching mechanism in the Bacillus subtilis yitJ S box/SAM-I riboswitch. J. Mol. Biol. 404, 803818 (2010).
  43. Wilson, R.C. et al. Tuning riboswitch regulation through conformational selection. J. Mol. Biol. 4, 926938 (2011).
  44. Ottink, O.M. et al. Ligand-induced folding of the guanine-sensing riboswitch is controlled by a combined predetermined induced fit mechanism. RNA 13, 22022212 (2007).
  45. 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, 52625273 (2007).
  46. 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, 91929197 (2010).
  47. Hermann, T. & Patel, D.J. Adaptive recognition by nucleic acid aptamers. Science 287, 820825 (2000).
  48. Dave, R., Terry, D.S., Munro, J.B. & Blanchard, S.C. Mitigating unwanted photophysical processes for improved single-molecule fluorescence imaging. Biophys. J. 96, 23712381 (2009).
  49. Qin, F. & Li, L. Model-based fitting of single-channel dwell-time distributions. Biophys. J. 87, 16571671 (2004).
  50. Lescoute, A. & Westhof, E. The interaction networks of structured RNAs. Nucleic Acids Res. 34, 65876604 (2006).

Download references

Author information

Affiliations

  1. Institute of Organic Chemistry, Center for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria.

    • Andrea Haller,
    • Ulrike Rieder,
    • Michaela Aigner &
    • Ronald Micura
  2. Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York, USA.

    • Scott C Blanchard

Contributions

A.H. performed RNA synthesis, labeling, enzymatic ligations and ensemble fluorescence spectroscopic measurements. S.C.B. performed smFRET measurements. U.R. performed NMR experiments. M.A. synthesized 5-fluorocytidine phosphoramidite for RNA synthesis. R.M., S.C.B. and A.H. analyzed the data. R.M. designed the study and wrote the paper (together with S.C.B. and A.H.).

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (2M)

    Supplementary Methods, Supplementary Figures 1–7 and Supplementary Table 1

Additional data