Coupling Green Fluorescent Protein Expression with Chemical Modification to Probe Functionally Relevant Riboswitch...

Noncoding RNAs engage in numerous biological activities including gene regulation. To fully understand RNA function it is necessary to probe biologically relevant conformations in living cells. To address this challenge, we coupled RNA-mediated regulation of the green fluorescent protein (GFP)uv-reporter gene to icSHAPE (in cell Selective 2'-Hydroxyl Acylation analyzed by Primer Extension). Our transcript-specific approach provides sensitive, fluorescence-based readout of the regulatory-RNA status as a means to coordinate chemical modification experiments. We chose a plasmid-based reporter compatible with Escherichia coli to allow use of knockout strains that eliminate endogenous effector biosynthesis. The approach was piloted using the Lactobacillus rhamnosus ( Lrh) preQ₁-II riboswitch, which senses the pyrrolopyrimidine metabolite preQ₁. Using an E. coli Δ queF strain incapable of preQ₁ anabolism, the Lrh riboswitch yielded nearly one log unit of GFPuv-gene repression resulting from exogenously added preQ₁. We then subjected cells in gene "on" and "off" states to icSHAPE. The resulting differential analysis indicated reduction in Lrh riboswitch flexibility in the P3 helix of the pseudoknot, which comprises the ribosome-binding site (RBS) paired with the anti-RBS. Such expression platform modulation was not observed by in vitro chemical probing and demonstrates that the crowded cellular environment does not preclude detection of compact and loose RNA-regulatory conformations. Here we describe the design, methods, interpretation, and caveats of Reporter Coupled (ReCo) icSHAPE. We also describe mapping of the differential ReCo-icSHAPE results onto the Lrh riboswitch-preQ₁ cocrystal structure. The approach should be readily applicable to functional RNAs triggered by effectors or environmental variations.