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 preQ riboswitch consists of a stem-loop and an adenine-rich single-stranded tail ("L3"), which adopt a pseudoknot structure upon binding of the ligand preQ . 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 preQ at all labeling positions tested, although modified and unmodified variants undergo the same global conformational changes at sufficiently high preQ 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 preQ riboswitch and that 2-AP substitution exerts complex and varied impacts on this riboswitch.