The Early Folding Intermediates of the Tetrahymena Ribozyme are Kinetically Trapped

Abstract The "RNA folding problem" is a fundamental and challenging question in contemporary biophysics. Understanding the mechanism(s) by which RNA molecules fold into compact structures capable of biological activity is important because RNA folding is closely tied to cellular regulation and metabolism and catalytic RNAs are potential reagents for gene therapy. Unlike the "protein folding problem" which has been under study for many decades, the study of RNA tertiary structure stability and folding is a relatively new field of endeavor. Thus, a detailed understanding of both the thermodynamics and kinetics of RNA folding are only now beginning to emerge. Kinetic traps have been observed in the late folding steps of the Tetrahymena ribozyme. In this study we extend our "synchrotron footprinting" analysis of the Tetrahymena ribozyme (Sclavi, et al. Science 279, 1940-1943, 1998) to probe the potential presence of kinetic traps in other steps in the folding mechanism. Examination of the folding in 3M urea demonstrates a significant increase in the rates of folding for early folding steps in the formation of the ribozyme tertiary structure. These data support the conclusion of Williamson and co-workers that the rate-limiting step in the folding of the Tetrahymena ribozyme is kinetically trapped by native interactions (Rook et al., J. Mol. Bio., 281, 609-620, 1998). Kinetic trapping also occurs in the formation of intermediates earlier in the folding reaction, and in these cases nonnative interactions may also play a role in the barrier to folding.