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

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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Figure 1: Structural depictions of the SAM-I riboswitch.
Figure 2: Mg2+- and ligand-dependent folding of SAM-I riboswitch.
Figure 3: Hidden Markov model (HMM) analysis of conformational dynamics of the SAM-I riboswitch.
Figure 4: Global fit analysis of smFRET efficiency histograms.
Figure 5: SAM-I riboswitch conformational states and their properties.

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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).

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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.

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Correspondence to G Ulrich Nienhaus.

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The authors declare no competing financial interests.

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Manz, C., Kobitski, A., Samanta, A. et al. Single-molecule FRET reveals the energy landscape of the full-length SAM-I riboswitch. Nat Chem Biol 13, 1172–1178 (2017). https://doi.org/10.1038/nchembio.2476

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