Probing and perturbing riboswitch folding using a fluorescent base analogue

Riboswitches are mRNA segments that regulate gene expression in response to ligand binding. The Class I preQ1 riboswitch consists of a stem-loop and an adenine-rich single-stranded tail (L3), which adopt a pseudoknot structure upon binding of the ligand preQ1. We inserted 2-aminopurine (2-AP), a fluorescent analogue of adenine (A), into the riboswitch at six different positions within L3. Here, 2-AP functions both as a spectroscopic probe and as a mutation that reveals how alteration of specific A residues impacts the riboswitch. Using fluorescence and circular dichroism spectroscopy, we found that 2-AP decreases the affinity of the riboswitch for preQ1 at all labeling positions tested, although modified and unmodified variants undergo the same global conformational changes at sufficiently high preQ1 concentration. 2-AP substitution is most detrimental to ligand binding at sites proximal to the ligand-binding pocket, while distal labeling sites exhibit the largest impacts on the stability of the L3 domain in the absence of ligand. Insertion of multiple 2-AP residues does not induce significant additional disruptions. Our results show that interactions involving the A residues in L3 play a critical role in ligand recognition by the preQ1 riboswitch and that 2-AP substitution exerts complex and varied impacts on this riboswitch. Fluorescent nucleic acid base analogues are popular probes of RNA and DNA structure. We placed the adenine analogue 2-aminopurine (2-AP) at various positions within a bacterial regulatory RNA and investigated its response to binding of a small molecule. Distinct local environments within this riboswitch yield distinct spectral properties of the probe and distinct changes upon ligand binding. 2-AP substitution reduces ligand affinity, highlighting the significance of interactions involving the native adenines.image

Automated summary

Riboswitches are mRNA segments that regulate gene expression in response to ligand binding. In this study, the insertion of 2-aminopurine (2-AP) into the riboswitch revealed how specific adenine residues impact the riboswitch and decrease its affinity for preQ1. The results highlight the critical role of A residues in ligand recognition and the complex effects of 2-AP substitution on the riboswitch.