Characterization of folding and misfolding of the Tetrahymena thermophila group I ribozyme

The functions of many cellular RNAs require that they fold into specific three-dimensional native structures, which typically involves arranging secondary structure elements and stabilizing the folded structure with tertiary contacts. However, RNA folding is inherently complex, as most RNAs fold along pathways containing multiple intermediates, including some misfolded intermediates that can accumulate and persist. Our understanding of the origins and structures of misfolded forms and the resolution of misfolding remains limited. Here, we investigate folding of the Tetrahymena intron, an extensively studied RNA folding model system since its initial discovery decades ago. The ribozyme variant predominantly misfolds, and slow refolding to the native state requires extensive structural disruption. Paradoxically, the misfolded conformation contains extensive native structure and lacks incorrect secondary and tertiary contacts despite requiring displacement of a native helix, termed P3, with incorrect secondary structure to misfold. We propose a model for a new origin of RNA misfolding to resolve this paradox, wherein misfolded ribozyme contains within its core incorrect arrangement of two single-stranded segments, i.e. altered topology. This model predicts a requirement for P3 disruption to exchange the misfolded and native topologies. We mutated P3 to modulate its stability and used the ribozyme's catalytic activity to show that P3 is disrupted during the refolding transition. Furthermore, we demonstrate that unfolding of the peripheral tertiary contacts precedes disruption of P3 to allow the necessary structural transitions. We then explored the influence of topology on the pathways leading to the misfolded and native states. Our results suggest that P3 exists in an earlier pathway intermediate that resembles the misfolded conformation, and that P3 unfolds to allow a small yet significant fraction of ribozyme to avoid misfolding. Despite being on a path to misfolding, the decision to misfold depends upon the probability of disrupting P3 and exchanging topology at this intermediate. Additionally, we show that having a stable P3 in the unfolded ribozyme allows almost complete avoidance of misfolding. Together, these studies lead to a physical model for folding and misfolding of a large RNA that is unprecedented in its scope and detail.