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Conformational capture of the SAM-II riboswitch

Nature Chemical Biology volume 7, pages 393–400 (2011)Cite this article

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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.

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Figure 1: Class-II SAM riboswitch.
Figure 2: Pseudoknot interaction analyzed by labeled SAM-II RNA using NMR and fluorescence spectroscopy.
Figure 3: Dynamics of pseudoknot formation of the SAM-II riboswitch analyzed by smFRET experiments.
Figure 4: SAM-II variants containing single 2-aminopurine labels: structural analysis and fluorescence response.
Figure 5: Thermodynamics and kinetics of the ligand-induced SAM-II riboswitch response.
Figure 6: Folding model of the SAM-II riboswitch.

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Acknowledgements

We thank D. Lafontaine for discussions on labeling of riboswitches for smFRET experiments; K. Lang (University of Innsbruck; presently at Medical Research Council, Cambridge) for a previous synthesis of 15N-labeled cytidine phosphoramidite; C. Kreutz and M. Tollinger for NMR assistance; D.S. Terry for contributions to the development and implementation of the single-molecule imaging platform and data analysis software used; and M. Soulière for critical reading of the manuscript. This work was funded by the Austrian Science Foundation FWF (I317 and P21641 to R.M.) and the Austrian Ministry of Science and Research (GenAU project consortium 'non-coding RNAs' P0726-012-012 to R.M.). S.C.B. was supported by the Irma T. Hirschl/Monique Weill-Caulier Trust.

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Authors and 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

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  1. Andrea Haller
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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.).

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Correspondence to Scott C Blanchard or Ronald Micura.

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Haller, A., Rieder, U., Aigner, M. et al. Conformational capture of the SAM-II riboswitch. Nat Chem Biol 7, 393–400 (2011). https://doi.org/10.1038/nchembio.562

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