Cobalamin Riboswitches Exhibit a Broad Range of Ability to Discriminate between Methylcobalamin and Adenosylcobalamin [Gene Regulation]

Riboswitches are a widely distributed class of regulatory RNAs in bacteria that modulate gene expression via small molecule induced conforma- tional changes. Generally, these RNA elements are grouped into classes based upon conserved primary and secondary structure and their cognate effector molecule. While this approach has been very suc- cessful at identifying new riboswitch families and defining their distributions, small sequence differ- ences between structurally related RNAs can al- ter their ligand selectivity and regulatory behav- ior. Herein, we use a structure-based mutagenic ap- proach to demonstrate that cobalamin riboswitches have a broad spectrum of preference for the two bio- logical forms of cobalamin in vitro using isothermal titration calorimetry. This selectivity is primarily mediated by the interaction between a peripheral element of the RNA that forms a T-loop module and a subset of nucleotides in the cobalamin-binding pocket. Cell-based fluorescence reporter assays in E. coli reveal mutations that switch effector prefer- ence in vitro lead to differential regulatory responses in a biological context. These data demonstrate that a more comprehensive analysis of representa- tive sequences of both previously and newly discov- ered classes of riboswitches might reveal subgroups of RNAs that respond to different effectors. Fur- thermore, this study demonstrates a second distinct means by which tertiary structural interactions in cobalamin riboswitches dictates ligand selectivity.

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