Structural and functional clues challenge the hypothesis that the yjdF riboswitch is natively regulated through broad recognition of azaaromatic compounds.

While most riboswitches are highly selective for their cognate ligand, the yjdF riboswitch is distinct in its ability to bind a broad set of aromatic compounds. This observation has led to the hypothesis that this RNA is regulated by toxic azaaromatic compounds, triggering a detoxification mechanism by activating translation of the YjdF protein in response to ligand binding. To understand how these compounds turn on gene expression by the yjdF riboswitch, we determined the crystal structure of the Bacillus subtilis yjdF riboswitch in complex with activating (chelerythrine) and nonactivating (lumichrome) ligands. These structures reveal that the RNA binds these compounds in a near-identical fashion, adopting the same local and global conformation. However, the unexpected extension of the regulatory helix through formation of several base pairs from highly conserved nucleotides suggests that this element plays an important role in ligand-dependent gene expression. Using a reporter assay in B. subtilis, we found that chelerythrine-dependent activation is insensitive to mutation of these conserved nucleotides that are essential for activation of the riboswitch. These data suggest that the yjdF riboswitch is responsive to a yet unknown cellular metabolite and remains an orphan riboswitch.