Abstract
Riboswitches are RNAs that recognize ligands and regulate gene expression. They are typically located in the untranslated region of bacterial messenger RNA and consist of an aptamer and an expression platform. In this study, we examine the folding pathway of the Vc2 (Vibrio cholerae) riboswitch aptamer domain, which targets the bacterial secondary messenger cyclic-di-GMP. We demonstrated by nuclear magnetic resonance (NMR) and isothermal titration calorimetry that the stable folding of the Vc2 riboswitch requires an adequate supply of Mg2+, Na+ and K+ ions. We found that Mg2+ has a crucial role in the pre-folding of the aptamer, while K+ is essential for establishing the long-range G-C interactions and stabilizing the ligand binding pocket. Precise imino proton assignments revealed the progressive folding of the aptamer. The results indicate that the P2 helix consists of weaker and more dynamic base pairs compared to the P1b helix, allowing the rearrangement of the base pairs in the P2 helix during the folding process required for effective ligand recognition. This study provides a profound understanding riboswitch architecture and dynamics at the atomic level under physiological conditions as well as structural information on apo-state RNA.