Corrin Ring Modifications Reveal the Chemical and Spatial Requirements for the B$_1$$_2$ $btuB$ Riboswitch Interaction

The btuB riboswitch is a regulatory RNA sequence controlling gene expression of the outer membrane B$_{12}$ transport protein BtuB by specifically binding coenzyme B$_{12}$ (AdoCbl) as its natural ligand. The B$_{12}$ sensing riboswitch class is known to accept various B$_{12}$ derivatives, leading to a division into two riboswitch subclasses, dependent on the size of the apical ligand. Here we focus on the role of side chains b and e on affinity and proper recognition, i. e. correct structural switch of the btuB RNA, which belongs to the AdoCbl‐binding class I. Chemical modification of these side chains disturbs crucial hydrogen bonds and/or electrostatic interactions with the RNA, its effect on both affinity and switching being monitored by in‐line probing. Chemical modifications at sidechain b of vitamin B$_{12}$ show larger effects indicating crucial B$_{12}$‐RNA interactions. When introducing the same modification to AdoCbl the influence of any side‐chain modification tested is reduced. This renders the impact of the adenosyl‐ligand for B$_{12}$‐btuB riboswitch recognition clearly beyond the known role in affinity.