UC San Diego

Riboswitches are 5'-untranslated regions of messenger RNA that bind a ligand and subsequently control bacterial gene expression. Riboswitches are comprised of a ligand-binding aptamer domain and an expression platform that controls transcription termination or affects translation initiation via controlling ribosome binding site accessibility. The widely conserved yybP-ykoY family of riboswitches bind manganese ions (Mn2+) to affect the expression of genes encoding Mn2+ transporter proteins. This dissertation explores the mechanism of one widespread Mn2+-sensing riboswitch, alx, in E. coli, which activates the translation of the Alx Mn2+ exporter. The alx riboswitch uniquely activates translation in vivo in response to both alkaline pH and increased Mn2+ concentration, making it a novel example of RNA-based gene regulation. Prior studies have determined the static structure and dynamics of isolated Mn2+-sensing aptamer domains. While valuable, these works did not include the riboswitch expression platform nor address why pH has a distinct effect on the alx riboswitch, but not on other yybP-ykoY family riboswitches. This dissertation addresses the question of how Mn2+ binding and cytoplasmic pH affect the structure and dynamics of the alx riboswitch in response to these two diverse cellular signals. Specifically, we employ a variety of techniques to study the structure, folding, and dynamics of the E. coli alx riboswitch. We use chemical probing techniques to understand how the structure and co-transcriptional folding of alx changes in response to Mn2+ and alkaline pH and compare these data to another E. coli Mn2+-sensing riboswitch, mntP, that does not exhibit a pH response in vivo. We use single-molecule FRET to understand how pH affects the alx aptamer dynamics and Mn2+-binding affinity. Finally, we employ in vivo lacZ gene reporter fusions to understand how these varying conditions affect gene expression in vivo and evaluate key mutants to determine the RNA elements that are responsible for the alx pH response. These data reveal that alkaline pH promotes a more open alx aptamer structure that is primed for sensing Mn2+ at lower concentrations compared to neutral pH. Additionally, we identify two key RNA loops that are responsible for the observed pH response. We also highlight that co-transcriptional folding and transcription kinetics are essential for the riboswitch to sense ligands and affect ribosome binding site accessibility. Given our observations, we present an antibiotic design strategy leveraging how co-transcriptional folding intermediate RNA elements could be targeted by antisense oligonucleotides in cells to inhibit the expression of genes essential for bacterial survival.