title: Tb3+-Cleavage Assays Reveal Specific Mg2+Binding Sites Necessary to Pre-fold thebtuBRiboswitch for AdoCbl Binding.
abstract: 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 thebtuBriboswitch fromE. 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 B12riboswitch ofS. thermophilumaided 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 thebtuBriboswitch indeed facilitates the assembly of the binding pocket needed for ligand recognition. Binding of the specific ligand, coenzyme B12(AdoCbl), to thebtuBaptamer 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.
authors.affiliation: Department of Chemistry, University of Zürich Zürich, Switzerland.
authors.forename: Pallavi K
authors.initials: PK
authors.lastname: Choudhary
authors.affiliation: Department of Chemistry, University of Zürich Zürich, Switzerland.
authors.forename: Sofia
authors.initials: S
authors.lastname: Gallo
authors.affiliation: Department of Chemistry, University of Zürich Zürich, Switzerland.
authors.forename: Roland K O
authors.initials: RK
authors.lastname: Sigel
keywords: Tb3+; coenzyme B12; metal ion binding; riboswitch; tertiary interactions