Multiple Unfolding Events during Native Folding of the Tetrahymena Group I Ribozyme

Despite the ubiquitous nature of misfolded intermediates in RNA folding, little is known about their physical properties or the folding transitions that allow them to continue folding productively. Folding of the Tetrahymena group I ribozyme includes sequential accumulation of two intermediates, termed Itrap and misfolded (M). Here, we probe the structure and folding transition of Itrap and compare them to those of M. Hydroxyl radical and dimethyl sulfate footprinting show that both Itrap and M are extensively structured and crudely resemble the native RNA. However, regions of the core P3–P8 domain are more exposed to solvent in Itrap than in M. Itrap rearranges to continue folding nearly 1000-fold faster than M, and urea accelerates folding of Itrap much less than M. Thus, the rate-limiting transition from Itrap requires a smaller increase in exposed surface. Mutations that disrupt peripheral tertiary contacts give large and nearly uniform increases in re-folding of M, whereas the same mutations give at most modest increases in folding from Itrap. Intriguingly, mutations within the peripheral element P5abc give 5- to 10-fold accelerations in escape from Itrap, whereas ablation of P13, which lies on the opposite surface in the native structure, near the P3–P8 domain, has no effect. Thus, the unfolding required from Itrap appears to be local, whereas the unfolding of M appears to be global. Further, the modest effects from several mutations suggest that there are multiple pathways for escape from Itrap and that escape is aided by loosening nearby native structural constraints, presumably to facilitate local movements of nucleotides or segments that have not formed native contacts. Overall, these and prior results suggest a model in which the global architecture and peripheral interactions of the RNA are achieved relatively early in folding. Multiple folding and re-folding events occur on the predominant pathway to the native state, with increasing native core interactions and cooperativity as folding progresses.