Structurally distinct manganese-sensing riboswitch aptamers regulate different expression platform architectures

Manganese (Mn)-sensing riboswitches protect bacteria from Mn toxicity by upregulating expression of Mn exporters. The Mn aptamers share key features but diverge in other important elements, including within the metal-binding core. Although X-ray crystal structures of isolated aptamers exist, these structural snapshots lack crucial details about how the aptamer communicates the presence or absence of ligand to the expression platform. In this work, we investigated the Mn-sensing translational riboswitches in Escherichia coli (mntP and alx), which differ in aptamer secondary structure, nucleotide sequence, and pH-dependence of Mn response. We performed co-transcriptional RNA chemical probing, allowing us to visualize RNA folding intermediates that form and resolve en route to the final folded riboswitch. For the first time, we report that sampling of metal ions by the RNA begins before the aptamer synthesis and folding are complete. At a single-nucleotide resolution, we pinpoint the transcription window where "riboswitching" occurs in response to Mn binding and uncover key differences in how the alx and mntP riboswitches fold. Finally, we describe riboswitch-specific effects of pH, providing insights into how two members of the same riboswitch family differentially sense two distinct environmental cues: concentration of Mn and pH.