The glycine riboswitch often occurs in a tandem architecture, with two ligand-binding domains (aptamers) followed by a single expression platform. Based on previous observations, we hypothesized that singlet versions of the glycine riboswitch, which contain only one aptamer domain, are able to bind glycine if appropriate structural contacts are maintained. An initial alignment of 17 putative singlet riboswitches indicated that the single consensus aptamer domain is flanked by a conserved peripheral stem-loop structure. These singlets were sorted into two subtypes based on whether the active aptamer domain precedes or follows the peripheral stem-loop, and an example of each subtype of singlet riboswitch was characterized biochemically. The singlets possess glycine-binding affinities comparable to those of previously published tandem examples, and the conserved peripheral domains form A-minor interactions with the single aptamer domain that are necessary for ligand-binding activity. Analysis of sequenced genomes identified a significant number of singlet glycine riboswitches. Based on these observations, we propose an expanded model for glycine riboswitch gene control that includes singlet and tandem architectures
