The widespread Mn2+-sensing yybP-ykoY riboswitch controls the expression of bacterial Mn2+ homeostasis genes. Here, we first determine the crystal structure of the ligand-bound yybP-ykoY riboswitch aptamer from Xanthomonas oryzae at 2.96 Å resolution, revealing two conformations with docked four-way junction (4WJ) and incompletely coordinated metal ions. In >100 µs of MD simulations, we observe that loss of divalents from the core triggers local structural perturbations in the adjacent docking interface, laying the foundation for signal transduction to the regulatory switch helix. Using single-molecule FRET, we unveil a previously unobserved extended 4WJ conformation that samples transient docked states in the presence of Mg2+. Only upon adding sub-millimolar Mn2+, however, can the 4WJ dock stably, a feature lost upon mutation of an adenosine contacting Mn2+ in the core. These observations illuminate how subtly differing ligand preferences of competing metal ions become amplified by the coupling of local with global RNA dynamics.

Department of Molecular Biology and Genetics Cornell University Ithaca NY 14850 USA

Department of Physical Chemistry Faculty of Science Palacký University tř 17 listopadu 12 Olomouc 771 46 Czech Republic

Institute of Biophysics of the Czech Academy of Sciences Kralovopolská 135 Brno 612 65 Czech Republic

Regional Centre of Advanced Technologies and Materials Faculty of Science Palacký University tř 17 listopadu 12 Olomouc 771 46 Czech Republic

Single Molecule Analysis Group and Center for RNA Biomedicine Department of Chemistry University of Michigan Ann Arbor MI 48109 USA

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