Requirements for efficient cotranscriptional regulatory switching in designed variants of the Bacillus subtilis pbuE adenine-responsive riboswitch

Riboswitches, generally located in the 5’-leader of bacterial mRNAs, direct expression via a small molecule-dependent structural switch informing 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 cotranscriptional context begun to be actively explored. To address this, we have performed a structure-guided mutagenic analysis of the B. subtilis pbuE adenine-responsive riboswitch, one of the simplest riboswitches containing a secondary structural switch. 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. Most importantly, it was found that local and long-range conformational dynamics in two regions of the aptamer domain have a significant effect upon efficient regulatory switching. Further, sequence features of the expression platform including the pre-aptamer leader sequence, a nucleation helix and a putative programmed pause have clear affects upon ligand-dependent regulation. 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.