<i>Escherichia coli</i> <i>yybP-ykoY</i> Riboswitch as a Tandem Riboswitch Regulated by Mn²⁺ and pH

The <i>Escherichia</i><i>coli</i> <i>yybP-ykoY</i> riboswitch regulates <i>mntP</i> and <i>alx</i> gene expression on the translation level. It contains two tandem domains regulated by Mn²⁺ and pH. This study investigates the tertiary structure and conformational dynamics of the <i>E. coli</i> <i>yybP-ykoY</i> riboswitch using a combination of crystallography, small-angle X-ray scattering (SAXS), and chemical probing. Our crystal structure of the aptamer domain at 3.8 Å reveals that the <i>yybP-ykoY</i> riboswitch aptamer domain forms a coaxial superhelix containing three helices connected by a three-way junction (3WJ), with L1 and L3 creating a pocket-like structure that binds Mg²⁺ and Mn²⁺. SHAPE probing and SAXS show that the <i>yybP-ykoY</i> riboswitch maintains a consistent conformation across pH conditions without Mn²⁺ but exhibits significant conformational changes under alkaline conditions when Mn²⁺ is present. These findings align with our proposed model, where Mn²⁺ binding induces a transition from an "OFF" to an "ON" state in alkaline conditions, while the Mn²⁺ remains bound to the aptamer independent of pH. This regulatory mechanism allows for more sophisticated control of gene expression, providing a finely tuned adaptive response to environmental changes.