Discrete structure of an RNA folding intermediate revealed by cryo-electron microscopy

Abstract

RNA folding occurs via a series of transitions between metastable intermediate states. It is unknown whether folding intermediates are discrete structures folding along defined pathways or heterogeneous ensembles folding along broad landscapes. We use cryo-electron microscopy and single-particle image reconstruction to determine the structure of the major folding intermediate of the specificity domain of a ribonuclease P ribozyme. Our results support the existence of a discrete conformation for this folding intermediate.

Figure 1. Cryo-EM and SPR of the S-domain intermediate structure

(A) Folding monitered by CD spectroscopy under conditions similar to cryo-EM studies. (B) Selected class averages of the intermediate compared with 2D projections from the 3D cryo-EM reconstruction. (C) SPR of the intermediate overlayed to an atomic model and secondary strucure. The final 3D map was generated using 60 class averages from ~11,600 particles.

Figure 2. Cryo-EM of S-domain intermediate with the helical extension on helix P9 (P9ext, green)

(A) P9ext (blue circles) has the same folding behavior as the wild-type S-domain (black line). (B) Cryo-EM reconstruction of the P9ext intermediate. The final 3D map was generated using 60 class averages from ~19,800 particles.