Cryo-EM reveals dynamics of Tetrahymena group I intron self-splicing

The group I self-splicing introns, like other RNAs and RNA–protein complexes, undergo multiple conformational changes in completing two transesterification reactions that cleave the intron and ligate the exons, but the detailed mechanism remains largely unknown. Here we use cryogenic electron microscopy to reveal six conformations associated with Tetrahymena intron self-splicing at 2.84–3.73 Å resolution directly following transcription, in which the RNAs can fold and splice cotranscriptionally. We identify two states with the dynamically undocked P1 helix in addition to the P1 docked conformation positioned for the first step, and three states associated with the second step, with one state carrying an unforeseen pseudoknotted structure collectively formed by the 5′-exon, 5′-intron and 3′-exon, providing an example of exons modulating splicing activity that is conserved among group IC1 introns. Translocations of nucleotides are observed in helix docking and intron splicing, whereas identification of metal ions validates the general two-metal-ion-splicing mechanism.