| Summary: | Riboswitches, generally located in the 5'-leader of bacterial mRNAs, direct expression via a small molecule-dependent structural switch that informs the transcriptional or translational machinery. While the structure and function of riboswitch effector-binding (aptamer) domains have been intensely studied, only recently have the requirements for efficient linkage between small molecule binding and the structural switch in the cellular and co-transcriptional context begun to be actively explored. To address this aspect of riboswitch function, we have performed a structure-guided mutagenic analysis of the B. subtilis pbuE adenine-responsive riboswitch, one of the simplest riboswitches that employs a strand displacement switching mechanism to regulate transcription. Using a cell-based fluorescent protein reporter assay to assess ligand-dependent regulatory activity in E. coli, these studies revealed previously unrecognized features of the riboswitch. Within the aptamer domain, local and long-range conformational dynamics influenced by sequences within helices have a significant effect upon efficient regulatory switching. Sequence features of the expression platform including the pre-aptamer leader sequence, a toehold helix and an RNA polymerase pause site all serve to promote strong ligand-dependent regulation. By optimizing these features, we were able to improve the performance of the B. subtilis pbuE riboswitch in E. coli from 5.6-fold induction of reporter gene expression by the wild type riboswitch to over 120-fold in the top performing designed variant. Together, these data point to sequence and structural features distributed throughout the riboswitch required to strike a balance between rates of ligand binding, transcription and secondary structural switching via a strand exchange mechanism and yield new insights into the design of artificial riboswitches.
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