Abstract

Riboswitches are structural RNA elements that are generally located in the 5′ untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform1,2,3. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time4. Here we use femtosecond X-ray free electron laser (XFEL) pulses5,6 to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of ‘mix-and-inject’ time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.

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Acknowledgements

Portions of this research were carried out at the Linac Coherent Light Source, a National User Facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The CXI instrument was funded by the LCLS Ultrafast Science Instruments (LUSI) project funded by the US Department of Energy, Office of Basic Energy Sciences. Use of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. We thank J. Strathern and M. Dunne for their support and S. Wakatsuki for discussions. This work is supported in part by the NSF-STC “BioXFEL” (NSF-1231306), the NIH Intramural Research Programs of NCI, CIT, NHLBI, and the US Department of Energy, Office of Biological and Environmental Research under Contract DE-AC02-06CH11357, the European Research Council, “Frontiers in Attosecond X-ray Science: Imaging and Spectroscopy (AXSIS)”, ERC-2013-SyG 609920, and the BMBF through project 05K16GU1.

Author information

Affiliations

  1. Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA

    • J. R. Stagno
    • , Y. Liu
    • , Y. R. Bhandari
    • , M. Swain
    • , D. R. Wendel
    • , R. A. Tuckey
    • , P. Yu
    • , M. Dyba
    • , S. G. Tarasov
    •  & Y.-X. Wang
  2. Department of Biochemistry, Arizona State University, Tempe, Arizona 85287, USA

    • C. E. Conrad
    • , J. D. Coe
    •  & P. Fromme
  3. Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA

    • C. E. Conrad
    • , J. D. Coe
    • , U. Weierstall
    • , P. Fromme
    • , N. A. Zatsepin
    •  & J. C. H. Spence
  4. Department of Biophysics, Johns Hopkins University, Baltimore, Maryland 21218, USA

    • S. Panja
    •  & S. A. Woodson
  5. Small Angle X-ray Scattering Core Facility, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA

    • L. Fan
  6. Department of Physics, Arizona State University, Tempe, Arizona 85287, USA

    • G. Nelson
    • , C. Li
    • , U. Weierstall
    • , N. A. Zatsepin
    •  & J. C. H. Spence
  7. Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestraße 85, 22607 Hamburg, Germany

    • T. A. White
    • , M. O. Wiedorn
    • , J. Knoska
    • , D. Oberthuer
    • , A. Barty
    •  & H. N. Chapman
  8. Department of Physics, University of Hamburg, Luruper Chaussee 149, 22607 Hamburg, Germany

    • M. O. Wiedorn
    • , J. Knoska
    •  & H. N. Chapman
  9. Hauptmann-Woodward Medical Research Institute, Buffalo, New York 14203, USA

    • T. D. Grant
  10. Center for Information Technology, National Institutes of Health, Bethesda, Maryland 20892-5624, USA

    • C. D. Schwieters
  11. Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

    • J. Zhang
  12. Laboratory of RNA Biophysics and Cellular Physiology, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

    • A. R. Ferré-D’Amaré
  13. Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, USA

    • D. E. Draper
  14. Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA

    • M. Liang
    • , M. S. Hunter
    •  & S. Boutet
  15. Structural Biology Center, Biosciences Division, Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 60439, USA

    • K. Tan
  16. X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 60439, USA

    • X. Zuo
  17. Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA

    • X. Ji

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Contributions

J.R.S. and Y.-X.W. designed experiments; Y.L. and P.Y. provided the RNA samples; J.R.S. and Y.L. crystallized the RNA; J.R.S., Y.L., Y.R.B., D.R.W., C.E.C., J.D.C., G.N., C.L., N.A.Z., M.O.W., D.O., J.K., T.D.G., M.S.H., S.B., M.L. and Y.-X.W. collected the SFX data; M.O.W., D.O., J.K. and H.N.C. designed the mixing setup; J.R.S., Y.R.B., D.R.W., T.A.W., A.B., R.A.T., N.A.Z., X.J. and T.D.G. processed and analysed the SFX data; J.R.S., Y.R.B., M.S., X.J. and Y.-X.W. interpreted the SFX data; N.A.Z., A.B., M.S.H., S.B., M.L., U.W., P.F., H.N.C. and J.C.H.S. contributed the XFEL expertise and support; Y.R.B., L.F., X.Z., C.D.S. and Y.-X.W. collected, analysed and interpreted SAXS data; J.R.S., C.E.C., K.T. and Y.-X.W. characterized samples; M.D. and Y.L. performed mass spectroscopy; S.G.T. and Y.L. performed ITC and fluorescence titration; S.P. and Y.L. collected and analysed binding data; X.Z. and S.A.W. modelled ligand binding kinetics; X.Z., S.P., S.A.W. and Y.-X.W. interpreted kinetic data; D.E.D., A.F.D. and J.Z. contributed discussions; J.R.S. and Y.-X.W. drafted the manuscript and all authors contributed to the revision.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Y.-X. Wang.

Reviewer Information Nature thanks J. Hajdu, R. Micura and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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https://doi.org/10.1038/nature20599

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