Characterization of the Tetrahymena Ribozyme Folding Pathway Using the Kinetic Footprinting Reagent Peroxynitrous Acid

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Abstract
Large RNAs fold into complex structures which determine their biological activities. A full understanding of both RNA structure and dynamics will include the description of the pathways by which these structures are formed. Kinetic footprinting [Sclavi, B., et al. (1997) J. Mol. Biol. 266, 144−159] has been shown to be a powerful method for the study of dynamic processes involving RNA. Here we describe the use of a readily available reagent, peroxynitrous acid, as a kinetic footprinting tool for the study of RNA folding. Hydroxyl radicals generated from this reagent were used to footprint the Tetrahymena ribozyme during its magnesium-dependent foldingin agreement with synchroton X-ray footprinting [Sclavi, B., et al. (1998) Science279, 1940−1943] and oligonucleotide/hybridization cleavage experiments [Zarrinkar, P. P., and Williamson, J. R. (1994) Science265, 918−924], this work suggests an ordered, hierarchical folding pathway for the ribozyme. Several slow steps in the folding pathway were observed in the peroxynitrous acid footprinting, but none of these corresponded to the rate-determining step of folding. This suggests that the formation of the global, protected structure is followed by one or more slow local rearrangements to yield the final active structure. These studies illustrate the utility of peroxynitrous acid as a reagent for the elucidation of RNA folding pathways and the study of RNA dynamics.