Tb³⁺-Cleavage Assays Reveal Specific Mg²⁺ Binding Sites Necessary to Pre-fold the btuB Riboswitch for AdoCbl Binding

Riboswitches are RNA elements that bind specific metabolites in order to regulate the gene expression involved in controlling the cellular concentration of the respective molecule or ion. Ligand recognition is mostly facilitated by Mg²⁺ mediated pre-organization of the riboswitch to an active tertiary fold. To predict these specific Mg²⁺ induced tertiary interactions of the btuB riboswitch from E. coli, we here report Mg²⁺ binding pockets in its aptameric part in both, the ligand-free and the ligand-bound form. An ensemble of weak and strong metal ion binding sites distributed over the entire aptamer was detected by terbium(III) cleavage assays, Tb³⁺ being an established Mg²⁺ mimic. Interestingly many of the Mⁿ⁺ (n = 2 or 3) binding sites involve conserved bases within the class of coenzyme B₁₂-binding riboswitches. Comparison with the published crystal structure of the coenzyme B₁₂ riboswitch of S. thermophilum aided in identifying a common set of Mⁿ⁺ binding sites that might be crucial for tertiary interactions involved in the organization of the aptamer. Our results suggest that Mⁿ⁺ binding at strategic locations of the btuB riboswitch indeed facilitates the assembly of the binding pocket needed for ligand recognition. Binding of the specific ligand, coenzyme B₁₂ (AdoCbl), to the btuB aptamer does however not lead to drastic alterations of these Mⁿ⁺ binding cores, indicating the lack of a major rearrangement within the three-dimensional structure of the RNA. This finding is strengthened by Tb³⁺ mediated footprints of the riboswitch's structure in its ligand-free and ligand-bound state indicating that AdoCbl indeed induces local changes rather than a global structural rearrangement.