A Large Collapsed-state RNA Can Exhibit Simple Exponential Single-molecule Dynamics Glenna J. Smith 1,4 , Kang Taek Lee 1,4 , Xiaohui Qu 2,4 , Zheng Xie 1,4 , Jelena Pesic 1,4 , Tobin R. Sosnick 3,4 , Tao Pan 3 and Norbert F. Scherer 1,4 ⁎ 1 Department of Chemistry, University of Chicago, Chicago, Illinois 60637, USA 2 Department of Physics, University of Chicago, Chicago, Illinois 60637, USA 3 Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA 4 Institute for Biophysical Dynamics, University of Chicago, Chicago, Illinois 60637, USA Received 8 December 2007; received in revised form 22 January 2008; accepted 23 January 2008 Available online 4 February 2008 The process of large RNA folding is believed to proceed from many collapsed structures to a unique functional structure requiring precise organization of nucleotides. The diversity of possible structures and stabilities of large RNAs could result in non-exponential folding kinetics (e.g. stretched exponential) under conditions where the molecules have not achieved their native state. We describe a single-molecule fluorescence resonance energy transfer (FRET) study of the collapsed-state region of the free energy landscape of the catalytic domain of RNase P RNA from Bacillus stearothermophilus (C thermo ). Ensemble measurements have shown that this 260 residue RNA folds cooperatively to its native state at ≥1 mM Mg 2+ , but little is known about the conformational dynamics at lower ionic strength. Our measurements of equilibrium conformational fluctuations reveal simple exponential kinetics that reflect a small number of discrete states instead of the expected inhomogeneous dynamics. The distribution of discrete dwell times, collected from an “ensemble” of 300 single molecules at each of a series of Mg 2+ concentrations, fit well to a double exponential, which indicates that the RNA conformational changes can be described as a four-state system. This finding is somewhat unexpected under [Mg 2+ ] conditions in which this RNA does not achieve its native state. Observation of discrete well-defined conformations in this large RNA that are stable on the seconds timescale at low [Mg 2+ ](b0.1 mM) suggests that even at low ionic strength, with a tremendous number of possible (weak) interactions, a few critical interactions may produce deep energy wells that allow for rapid averaging of motions within each well, and yield kinetics that are relatively simple. © 2008 Elsevier Ltd. All rights reserved. Edited by D. E. Draper Keywords: FRET; single molecule; RNA folding; collapsed state Introduction RNA folding is structurally hierarchical due to three effects: the stacking of bases facilitated by base-pair hydrogen bonding, the electrostatic screening of the polyanionic backbone by mono-or divalent cations, and specific binding of cations. 1,2 Sequestering base pairs in local runs of helical se- condary structure occurs very early (i.e., at low ionic strength) in the process of RNA folding. 3–5 Tertiary compaction into dense functional structures requires much higher concentrations of Mg 2+ for sufficient screening of the phosphate groups. 6 Because sec- ondary structure can be stable independent of ter- tiary structure, RNA passes through collapsed *Corresponding author. Department of Chemistry, University of Chicago, Chicago, Illinois 60637, USA. E-mail address: nfschere@uchicago.edu. Present address: K. T. Lee, Korea Research Institute of Chemical Technology, Daejeon 305-343, Korea. Abbreviations used: C thermo L18, catalytic domain of RNase P RNA from Bacillus stearothermophilus labeled with a FRET pair on the 3′ end and L18 loop; FRET, fluorescence resonance energy transfer; E FRET , efficiency of resonance energy transfer. doi:10.1016/j.jmb.2008.01.078 J. Mol. Biol. (2008) 378, 941–951 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.