Two riboswitches that share a common ligand-binding fold show dramatic differences in the ability to accommodate mutations

Riboswitches are structured RNA elements that sense small molecules, leading conformations that control expression of downstream genes. Prequeuosine1 (preQ1)-sensing riboswitches are a widespread family of bacterial regulatory elements that fall into three classes (I, II & III). The class II and III riboswitches adopt different overall pseudoknot folds that allow binding of a single preQ1 metabolite within a three-way-helical junction. Despite the absence of sequence homology, both riboswitches use ten identical nucleotides for preQ1 recognition. Previous analysis of the preQ1-II (class II) riboswitch showed a high sensitivity to core mutations, which severely impaired both preQ1 affinity and gene-regulation. Here, we made homologous mutations to the preQ1-III riboswitch. We determined co-crystal structures of A52G, A84G, D84 and U8C/A85G mutants to 2.55-3.04 A resolution. The mutant structures show a remarkable resilience in their ability to form compensatory hydrogen bonds and backbone conformational changes. Chemical modification analysis shows localized increases in RNA flexibility for each preQ1-III mutant compared to the wildtype. Molecular dynamics simulations suggest mutations are not destabilizing. Indeed, analysis of preQ1 affinity showed that mutants maintain relatively tight binding (KD range of 9.8-38 nM) compared to homologous preQ1-II mutations (KD range of 62-518 nM). Analysis of gene-regulatory function in live cells showed that preQ1-III mutations maintained significant function (EC50 range of 82 nM to 1.1 uM) relative to preQ1-II mutations that impair gene regulation (EC50 range of 28 uM to 182 uM). Overall, the results demonstrate that the impact of riboswitch mutations depends on their context within the overall RNA fold.