Riboswitches are structured mRNA sequences that regulate gene expression by directly binding intracellular metabolites. Generating the appropriate regulatory response requires the RNA rapidly and stably acquire higher-order structure to form the binding pocket, bind the appropriate effector molecule and undergo a structural transition to inform the expression machinery. These requirements place riboswitches under strong kinetic constraints, likely restricting the sequence space accessible by recurrent structural modules such as the kink turn and the T-loop. Class-II cobalamin riboswitches contain two T-loop modules: one directing global folding of the RNA and another buttressing the ligand binding pocket. While the T-loop module directing folding is highly conserved, the T-loop associated with binding is substantially less so, with no clear consensus sequence. To further understand the functional role of the binding-associated module, a functional genetic screen of a library of riboswitches with the T-loop and its interacting nucleotides was used to build an experimental phylogeny comprised of sequences that possess a wide range of cobalamin-dependent regulatory activity. Our results reveal conservation patterns of the T-loop and its interaction with the binding core that allow for rapid tertiary structure formation and demonstrate its importance for generating strong ligand-dependent repression of mRNA expression.

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.