Identification of MetaboliteRiboswitch Interactions Using Nucleotide Analog Interference Mapping and Suppression

Riboswitches are RNA elements capable of modulating gene expression through interaction with cellular metabolites. One member of the riboswitch family, the glmS riboswitch, is unique among riboswitches in that it modulates gene expression by undergoing self-cleavage in the presence of its metabolite glucosamine-6-phosphate (GlcN6P). In order to investigate the interactions between the glmS RNA and GlcN6P we performed nucleotide analog interference mapping (NAIM) and suppression (NAIS). These techniques have been previously used to identify important functional groups in and tertiary contacts necessary for self-splicing and self-cleaving by catalytic RNAs, RNA–protein complexes, RNA folding, and RNA–metal ion interactions. Described here are the details of NAIM and NAIS experiments we have utilized to investigate RNA–ligand interactions between the glmS riboswitch and GlcN6P. These techniques can be employed to study a wide variety of RNA–small molecule interactions.