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.

Topological crossing in the misfolded Tetrahymena ribozyme resolved by cryo-EM

“…Here, taking advantage of the ability of single-particle cryo-EM to computationally sort out highly heterogeneous or flexible samples ( 22 – 24 ), we obtained 3D structures of M and N states of Tetrahymena ribozyme from a single specimen, revealing the strand topology and global architecture of M. We provide direct structural evidence that M has a partially native-like core and extensively native-like periphery, except for local rearrangements at the catalytic core involving a rotated P7 helix connected to two single-stranded segments, J7/3 and J8/7, that are crossed ( Movie S3 ). An independent cryo-EM analysis of M and N has been reported as a preprint simultaneously with the preparation of this manuscript; though at lower resolution, those completely independent data further support our structures ( 25 ).…”

Combining simulations and experiments to investigate RNA dynamics

“…Cryo-electron-microscopy experiments have been used in so-called flexible-fitting MD simulations [32,33] to refine structures [34,35]. In this setting, MD simulations are used as a modeling tool to generate structures compatible with experiments, but the quantitative degree of dynamics compatible with the experimental density maps is not explicitly controlled.…”