Fig. 7. Revised group II intron splicing cycle.
a Snapshots of the intron active site derived from the crystal structures and the MD simulations. In the pre-catalytic state, which corresponds to the pre-hydrolytic state of the first step of splicing, a water molecule (light blue sphere, W) or the 2′-OH of a bulged adenosine are poised for nucleophilic attack on the scissile phosphate (orange sphere, SP) (PDB id. 4FAQ). In this state, the intron established the K1–N7G288 interaction, and the nucleophile cleaves the intron-5en junction. Immediately after hydrolytic cleavage, the proton released by the nucleophile initially to the bulk solvent is shuttled to C358 in the catalytic triad. C358 protonation favors release of the metal ions cluster and toggling of J2/3 junction (PDB id. 4FAX). This conformational rearrangement likely prompts the release of the products of the first step of splicing, subsequent rearrangement of D6, and reverse toggling of J2/3 to reconstitute the active site and align second step reactants. Finally, cleavage of intron-3e junction leads to the release of free linear intron (PDB id. 4E8M) and spliced exons. The free intron is still an active ribozyme, which can retrotranspose into target genomic DNA and re-initiate a new splicing cycle (dashed gray arrow). Relevant intron motifs are shown as cartoon representations in green. The 5e is in blue, the 3e in brown. K1 is shown as a violet sphere, M1 and M2 as yellow spheres. K2 is not shown for clarity. Intron states described in this work are labeled red, and differences between consecutive panels are indicated as bold orange labels. b Sketches of the intron active sites corresponding to the precatalytic state (with K1–N7 interaction, left) and to the K1 release and toggling (right) drawn in the same style as in Fig. 1a. For clarity, only the 5′-splice site is represented. The red dotted arrow indicates the proton transfer pathway from the nucleophile (Nuc) to N3C358.