Probing RNA Conformational Equilibria within the Functional Cellular Context

Summary: Low-abundance short-lived non-native conformations referred to as excited states (ESs) are increasingly observed in vitro and implicated in the folding and biological activities of regulatory RNAs. We developed an approach for assessing the relative abundance of RNA ESs within the functional cellular context. Nuclear magnetic resonance (NMR) spectroscopy was used to estimate the degree to which substitution mutations bias conformational equilibria toward the inactive ES in vitro. The cellular activity of the ES-stabilizing mutants was used as an indirect measure of the conformational equilibria within the functional cellular context. Compensatory mutations that restore the ground-state conformation were used to control for changes in sequence. Using this approach, we show that the ESs of two regulatory RNAs from HIV-1, the transactivation response element (TAR) and the Rev response element (RRE), likely form in cells with abundances comparable to those measured in vitro, and their targeted stabilization may provide an avenue for developing anti-HIV therapeutics. : Ganser et al. use point mutations to bias conformational equilibria in HIV-1 TAR and RRE RNA toward inactive excited states. Comparing the conformational bias induced in vitro with biological activity measured in cells suggests similar structural dynamics in vitro and in cells for these two RNAs. Keywords: HIV-1 transactivation response element, TAR, Rev response element, RRE, RNA dynamics, RNA drug discovery, transactivation, excited state, RNA structure, structure mapping, cellular activity, conformational switches