Magnesium Mediated Fluoride Binding Anchors the Docked Conformation of the Fluoride Riboswitch

Abstract

Riboswitches are structured non-coding RNA elements typically found in the 5' untranslated regions of bacterial mRNAs. They regulate gene expression, most often at the level of transcription and translation, through the binding of specific ligands that induce changes in the riboswitch structure. Fluoride riboswitches are a family of transcriptional riboswitches that are present in many bacteria and archaea; regulate transcription by preventing the formation of an intrinsic terminator in presence of fluoride. Previous studies have shed light on its structure and the importance of the transcriptional machinery for riboswitch folding, a deep conformational analysis at different points during transcription are needed to fully understand the impact of ligand-binding during transcription. Therefore, we explored the conformational dynamics of the Bacillus cereus crcB fluoride riboswitch at the single-molecule level for both isolated RNA and active transcription elongation complexes. We resolved three conformational states: two undocked and one pseudoknot-structured docked state, and determined their fractional population and interconversion rates. The first undocked state was observed at early stage of transcription; while the other two states, a docked and an undocked state, appear when the aptamer is fully transcribed by RNA polymerase. The riboswitch folds into a transient dynamic docked conformation in presence of magnesium, which upon further binding of fluoride folds into a stably docked conformation. This feature is lost upon disrupting a long-range base pair A40-U48. In active transcription elongation complexes, RNA polymerase fine-tunes the activation energy and promotes the riboswitch to fold into the docked conformation. Further, we showed that ligand binding at short transcript lengths is an early step on the pathway to the docked conformation. These results demonstrate a multi-step mechanism of magnesium- and ligand-dependent conformational transitions that enable regulation of downstream gene expression.