A Kissing Loop Is Important for btuB Riboswitch Ligand Sensing and Regulatory Control

RNA-based genetic regulation is exemplified by metabolite-binding riboswitches that modulate gene expression through conformational changes. Crystal structures show that the Escherichia coli btuB riboswitch contains a kissing loop interaction that is in close proximity to the bound ligand. To analyze the role of the kissing loop interaction in the riboswitch regulatory mechanism, we used RNase H cleavage assays to probe the structure of nascent riboswitch transcripts produced by the E. coli RNA polymerase. By monitoring the folding of the aptamer, kissing loop, and riboswitch expression platform, we established the conformation of each structural component in the absence or presence of bound adenosylcobalamin. We found that the kissing loop interaction is not essential for ligand binding. However, we showed that kissing loop formation improves ligand binding efficiency and is required to couple ligand binding to the riboswitch conformational changes involved in regulating gene expression. These results support a mechanism by which the btuB riboswitch modulates the formation of a tertiary structure to perform metabolite sensing and regulate gene expression. Background: The btuB riboswitch contains an unusual kissing loop structure that exhibits few interactions with bound adenosylcobalamin. Results: Structural probing and in vivo analysis show that kissing loop mutations strongly uncouple ligand binding from riboswitch regulation. Conclusion: The kissing loop is pivotal for coordinating riboswitch conformational changes. Significance: In contrast to most riboswitches, btuB relies on a kissing loop to achieve both metabolite sensing and gene regulation.