Cryo-EM reveals an entangled kinetic trap in the folding pathway of a catalytic RNA

Abstract

Functional RNAs fold through complex pathways that can contain misfolded "kinetic traps." A complete model of RNA folding requires understanding the formation of such misfolded states, but they are difficult to characterize due to their transient and potentially conformationally dynamic nature. We used cryo-electron microscopy (cryo-EM) to visualize a long-lived misfolded state in the folding pathway of the Tetrahymena thermophila group I intron, a paradigmatic RNA structure-function model system. The structure revealed how this state forms native-like secondary structure and tertiary contacts but contains two incorrectly crossed strands, consistent with a previous model. This incorrect topology mispositions a critical catalytic domain and cannot be resolved locally, as extensive refolding is required. This work provides a structural framework for interpreting decades of biochemical and functional studies and demonstrates the power of cryo-EM for the exploration of RNA folding pathways.