UC San Diego

Riboswitches are 5’-untranslated regions of a messenger RNA that control the fate of their respective transcript by changing their conformation in response to a ligand-binding event, effectively turning the gene “on” or “off”. This dissertation investigates two riboswitches in E. coli that sense manganese (Mn), which upon Mn binding, rearrange their folded structures into a conformation that is accessible for ribosome binding and subsequent translation of the downstream genes, alx and mntP. While prior study uncovered the structure and dynamics of Mn-sensing riboswitch aptamers, it remains unknown how Mn binding rearranges the full-length riboswitch into a translationally active conformation. Further, prior work missed the aspect of active transcription that is critical for riboswitch folding and ligand-sensing. To address these gaps in knowledge, we employed a suite of biochemical techniques to elucidate how the E. coli Mn-sensing riboswitches, alx and mntP, fold and respond to Mn binding co-transcriptionally. First, we have applied in vitro transcription methods to study the kinetics of RNA polymerase (RNAP) transcription on the alx and mntP riboswitch sequences. Specifically, we characterized transcriptional pauses encountered by RNAP during synthesis of the alx and mntP riboswitches, which may coordinate key RNA folding transitions. Next, we explored how the alx and mntP RNAs fold co-transcriptionally using RNA chemical probing methods. These data uncovered that the alx and mntP riboswitch aptamer folding intermediates can sense Mn, challenging the notion that a fully folded riboswitch aptamer is required for ligand sensing. Further, we provide the first insights into how the conserved alx and mntP aptamer domains “communicate” with their expression platforms, which diverge in sequence and in structure. Lastly, we show that specific differences in alx and mntP aptamer sequence may explain how pH differentially tunes Mn affinity for the riboswitch aptamers. To underscore the importance of riboswitch co-transcriptional folding, we highlight how RNA folding intermediates may be targeted by antisense oligonucleotides (ASOs) – an exciting new strategy for riboswitch inhibitor design.