The evolution of RNA conformation with Mg2+ concentration ([Mg2+]) is typically determined from equilibrium titration measurements or nonequilibrium single [Mg2+]-jump measurements. We study the folding of single RNA molecules in response to a series of periodic [Mg2+] jumps. The 260-residue catalytic domain of RNase P RNA from Bacillus stearothermophilus is immobilized in a microfluidic flow chamber, and the RNA conformational changes are probed by fluorescence resonance energy transfer (FRET). The kinetics of population redistribution after a [Mg2+] jump and the observed connectivity of FRET states reveal details of the folding pathway that complement and transcend information from equilibrium or single-jump measurements. FRET trajectories for jumps from [Mg2+] = 0.01 to 0.1 mM exhibit two-state behavior whereas jumps from 0.01 mM to 0.4 mM exhibit two-state unfolding but multistate folding behavior. RNA molecules in the low and high FRET states before the [Mg2+] increase are observed to undergo dynamics in two distinct regions of the free energy landscape separated by a high barrier. We describe the RNA structural changes involved in crossing this barrier as a “hidden” degree of freedom because the changes do not alter the detected FRET value but do alter the observed dynamics. The associated memory prevents the populations from achieving their equilibrium values at the end of the 5- to 10-sec [Mg2+] interval, thereby creating a nonequilibrium steady-state condition. The capability of interrogating nonequilibrium steady-state RNA conformations and the adjustable period of [Mg2+]-jump cycles makes it possible to probe regions of the free energy landscape that are infrequently sampled in equilibrium or single-jump measurements
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