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Single-molecule FRET reveals the energy landscape of the full-length SAM-I riboswitch

Nature Chemical Biology volume 13, pages 11721178 (2017) | Download Citation

Abstract

S-adenosyl-L-methionine (SAM) ligand binding induces major structural changes in SAM-I riboswitches, through which gene expression is regulated via transcription termination. Little is known about the conformations and motions governing the function of the full-length Bacillus subtilis yitJ SAM-I riboswitch. Therefore, we have explored its conformational energy landscape as a function of Mg2+ and SAM ligand concentrations using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling analysis. We resolved four conformational states both in the presence and the absence of SAM and determined their Mg2+-dependent fractional populations and conformational dynamics, including state lifetimes, interconversion rate coefficients and equilibration timescales. Riboswitches with terminator and antiterminator folds coexist, and SAM binding only gradually shifts the populations toward terminator states. We observed a pronounced acceleration of conformational transitions upon SAM binding, which may be crucial for off-switching during the brief decision window before expression of the downstream gene.

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Acknowledgements

We thank A. Schug (KIT) for fruitful discussions. This work was supported by grants from the Karlsruhe Heidelberg Research Partnership (HEiKA) and the Volkswagen Foundation (grant 82549) to A.J. and G.U.N. G.U.N. was also funded by the Helmholtz program Science and Technology of Nanosystems (STN), Karlsruhe School of Optics and Photonics (KSOP) and the Deutsche Forschungsgemeinschaft (DFG), grant GRK 2039. B.G.K. was funded by the DFG through grant CRC 1114 (project B05).

Author information

Affiliations

  1. Institute of Applied Physics, Karlsruhe Institute of Technology, Karlsruhe, Germany.

    • Christoph Manz
    • , Andrei Yu Kobitski
    •  & G Ulrich Nienhaus
  2. Heidelberg Karlsruhe Research Partnership (HEiKA), Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany.

    • Christoph Manz
    • , Andres Jäschke
    •  & G Ulrich Nienhaus
  3. Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, Heidelberg, Germany.

    • Ayan Samanta
    •  & Andres Jäschke
  4. Freie Universität Berlin, Institute of Chemistry and Biochemistry, Berlin, Germany.

    • Bettina G Keller
  5. Institute of Nanotechnology and Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany.

    • G Ulrich Nienhaus
  6. Department of Physics, University of Illinois at Urbana–Champaign, Urbana, Illinois, USA.

    • G Ulrich Nienhaus

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Contributions

G.U.N. and A.J. designed research; A.S. synthesized RNA constructs; C.M. and A.Y.K. built the experimental setup and took data; C.M., A.Y.K. and B.G.K. analyzed data and all authors contributed to manuscript writing.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to G Ulrich Nienhaus.

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DOI

https://doi.org/10.1038/nchembio.2476