Decoding the Complex Functional Landscape of the <i>ykkC</i> Riboswitches

The <i>ykkC</i> class is the most diverse riboswitch class to date, recognizing structurally and chemically diverse ligands using only minor changes in sequence and structure. Structural studies have demonstrated how sequence changes correspond to altered specificity; however, they are insufficient to define the requirements for functional riboswitch specificity. Here, we report an extensive mutational analysis of the ppGpp riboswitch to investigate the functional role in transcriptional control for this variant riboswitch. Disruption of the terminator hairpin at a single base pair is sufficient to abolish nearly all function, highlighting the fine-tuning of the terminator hairpin to its corresponding aptamer domain. This fine-tuning has been observed in other riboswitches, suggesting that high levels of tunability may be a common feature of riboswitches. Additionally, mutational analysis shows that the previously reported binding site position, G93, does not necessarily correspond to PRPP-driven function as expected. Phylogenetic analysis of natural riboswitches that contain G93 revealed an additional <i>ykkC</i> subclass that binds to both XMP and GMP. This variant subclass is associated with genes for <i>de novo</i> GMP synthesis. Identification of this variant class provides further evidence for small sequence changes corresponding to altered ligand specificity.