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Three-state mechanism couples ligand and temperature sensing in riboswitches

Abstract

Riboswitches are cis-acting gene-regulatory RNA elements that can function at the level of transcription, translation and RNA cleavage1,2,3. The commonly accepted molecular mechanism for riboswitch function proposes a ligand-dependent conformational switch between two mutually exclusive states4. According to this mechanism, ligand binding to an aptamer domain induces an allosteric conformational switch of an expression platform, leading to activation or repression of ligand-related gene expression5. However, many riboswitch properties cannot be explained by a pure two-state mechanism. Here we show that the regulation mechanism of the adenine-sensing riboswitch, encoded by the add gene on chromosome II of the human Gram-negative pathogenic bacterium Vibrio vulnificus6, is notably different from a two-state switch mechanism in that it involves three distinct stable conformations. We characterized the temperature and Mg2+ dependence of the population ratios of the three conformations and the kinetics of their interconversion at nucleotide resolution. The observed temperature dependence of a pre-equilibrium involving two structurally distinct ligand-free conformations of the add riboswitch conferred efficient regulation over a physiologically relevant temperature range. Such robust switching is a key requirement for gene regulation in bacteria that have to adapt to environments with varying temperatures. The translational adenine-sensing riboswitch represents the first example, to our knowledge, of a temperature-compensated regulatory RNA element.

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Figure 1: Modulation of the conformational equilibrium of the wild-type adenine-sensing riboswitch by temperature, Mg2+ and cognate ligand.
Figure 2: Comparison of ligand binding kinetics of WT and MutP2 adenine-sensing riboswitch constructs.
Figure 3: Adenine-dependent expression regulation is only detected for the wild-type riboswitch.
Figure 4: Ligand-dependent conformational equilibria and simulation of the switching efficiency.

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Acknowledgements

We thank E. Stirnal, H. Keller and C. Richter for technical support. We thank F. Narberhaus, J. Wöhnert, J. Wachtveitl, J. Soppa, E. Schleiff, A. Heckel, M. Hengesbach and C. Griesinger for stimulating discussions. This work was funded by the German funding agency (DFG) in Collaborative Research Center 902.

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A.R., S.N., K.S., F.B. and B.F. conducted experiments. All authors contributed to the analysis of the data and the writing of the manuscript.

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Correspondence to Boris Fürtig or Harald Schwalbe.

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The authors declare no competing financial interests.

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Reining, A., Nozinovic, S., Schlepckow, K. et al. Three-state mechanism couples ligand and temperature sensing in riboswitches. Nature 499, 355–359 (2013). https://doi.org/10.1038/nature12378

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