Probing and perturbing riboswitch folding using a fluorescent base analogue

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. AbstractRiboswitches 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...
Source: Photochemistry and Photobiology - Category: Science Authors: Tags: SPECIAL ISSUE RESEARCH ARTICLE Source Type: research