Single-molecule studies highlight conformational heterogeneity in the early folding steps of a large ribozyme.
The equilibrium folding of the catalytic domain of Bacillus subtilis RNase P RNA is investigated by single-molecule fluorescence resonance energy transfer (FRET). Previous ensemble studies of this 255-nucleotide ribozyme described the equilibrium folding with two transitions, U-to-I(eq)-to-N, and focused on the I(eq)-to-N transition. The present study focuses on the U-to-I(eq) transition. Comparative ensemble measurements of the ribozyme construct labeled with fluorescein at the 5' end and Cy3 at the 3' end show that modifications required for labeling do not interfere with folding and help to define the Mg(2+) concentration range for the U-to-I(eq) transition. Histogram analysis of the Mg(2+)-dependent single-molecule FRET efficiency reveals two previously undetermined folding intermediates. The single-molecule FRET trajectories exhibit non-two-state and nonergodic behaviors at intermediate Mg(2+) concentrations on the time scale of seconds. The trajectories at intermediate Mg(2+) concentrations are classified into five classes based on three FRET levels and their dynamics of interconversion within the measured time range. This heterogeneity, together with the observation of "nonsudden jump" FRET transitions, indicates that the early folding steps of this ribozyme involve a series of intermediates with different degrees of kinetic isolation and that folding occurs under kinetic control and involves many "local" conformational switches. A free energy contour is constructed to illustrate the complex folding surface.
Xie
Zheng
Z
Institute for Biophysical Dynamics and Department of Chemistry, University of Chicago, Chicago, IL 60637, USA.
Srividya
Narayanan
N
Sosnick
Tobin R
TR
Pan
Tao
T
Scherer
Norbert F
NF
D002942:Circular Dichroism; D004735:Energy Transfer; D005453:Fluorescence; D011487:Protein Conformation; D017510:Protein Folding; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*
D016337:RNA, Catalytic