The rate-limiting step in the folding of a large ribozyme without kinetic traps.
A fundamental question in RNA folding is the nature of the rate-limiting step. Folding of large RNAs often is trapped by the need to undo misfolded structures, which precludes the study of the other, potentially more interesting aspects in the rate-limiting step, such as conformational search, metal ion binding, and the role of productive intermediates. The catalytic domain of the Bacillus subtilis RNase P RNA folds without a kinetic trap, thereby providing an ideal system to elucidate these steps. We analyzed the folding kinetics by using fluorescence and absorbance spectroscopies, catalytic activity, and synchrotron small-angle x-ray scattering. Folding begins with the rapid formation of early intermediates wherein the majority of conformational search occurs, followed by the slower formation of subsequent intermediates. Before the rate-limiting step, more than 98% of the total structure has formed. The rate-limiting step is a small-scale structural rearrangement involving prebound metal ions.
Fang
X-W
XW
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA.
Thiyagarajan
P
P
Sosnick
T R
TR
Pan
T
T
D001483:Base Sequence; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017510:Protein Folding; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D008670:Metals; D016337:RNA, Catalytic