Visualizing a two-state conformational ensemble in stem-loop 3 of the transcriptional regulator 7SK RNA.

Structural plasticity is integral to RNA function; however, there are currently few methods to quantitatively resolve RNAs that have multiple structural states. NMR spectroscopy is a powerful approach for resolving conformational ensembles but is size-limited. Chemical probing is well-suited for large RNAs but provides limited structural and no kinetics information. Here, we integrate the two approaches to visualize a two-state conformational ensemble for the central stem-loop 3 (SL3) of 7SK RNA, a critical element for 7SK RNA function in transcription regulation. We find that the SL3 distal end exchanges between two equally populated yet structurally distinct states in both isolated SL3 constructs and full-length 7SK RNA. We rationally designed constructs that lock SL3 into a single state and demonstrate that both chemical probing and NMR data fit to a linear combination of the two states. Comparison of vertebrate 7SK RNA sequences shows conservation of both states, suggesting functional importance. These results provide new insights into 7SK RNA structural dynamics and demonstrate the utility of integrating chemical probing with NMR spectroscopy to gain quantitative insights into RNA conformational ensembles.