Abstract
7SK is a conserved noncoding RNA that regulates transcription by sequestering the transcription factor P-TEFb. 7SK function entails complex changes in RNA structure, but characterizing RNA dynamics in cells remains an unsolved challenge. We developed a single-molecule chemical probing strategy, DANCE-MaP (deconvolution and annotation of ribonucleic conformational ensembles), that defines per-nucleotide reactivity, direct base pairing interactions, tertiary interactions, and thermodynamic populations for each state in RNA structural ensembles from a single experiment. DANCE-MaP reveals that 7SK RNA encodes a large-scale structural switch that couples dissolution of the P-TEFb binding site to structural remodeling at distal release factor binding sites. The 7SK structural equilibrium shifts in response to cell growth and stress and can be targeted to modulate expression of P-TEFbresponsive genes. Our study reveals that RNA structural dynamics underlie 7SK function as an integrator of diverse cellular signals to control transcription and establishes the power of DANCE-MaP to define RNA dynamics in cells.