A transient third metal ion shapes the catalytic cycle of a group I intron ribozyme
Journal:
bioRxiv
Published Date:
Oct 9, 2026
Abstract
The self-splicing group I intron ribozyme from Tetrahymena thermophillia (THr) is a prototypical example of how RNA overcomes its limited chemical repertoire to form complex, negatively charged junctions and catalyze chemical reactions. Despite over 30 years of biochemical and structural characterization, questions still remain regarding the requirements for THr catalysis that may inform structure based drug design against group I introns, as well as the use of THr as an RNA editing tool. Recent cryo-EM structures have provided an unprecedented window into the first and second steps of splicing, enabling detailed mechanistic investigation at the atomistic level. In this work, we leverage solvation theory, molecular dynamics, alchemical free energy simulations, and machine learning accelerated QM/MM to probe outstanding questions related to 1) the role of a possible third Mg2+ binding in the active site, 2) differences in catalytic requirements between Gexo cofactors and between the first and second steps of splicing, 3) the potential role of an acid or base, and 4) restrictions on the identity of the 5' exon splice site nucleotide, U(-1). We propose that a transient, cofactor dependent, interaction with a third metal participates in the first step, while a higher affinity interaction with this third ion is required in the second step. We also propose that the presence of a phosphate on the Gexo in the first step, and analogously the G{omega} in the second step, forms a metal ion binding site not present when guanosine is bound. Finally, we propose and computationally test design modifications of G22 (isofunctional substitutions of 2,6-diaminopurine that tune the pKa at the N1 position) that we predict will enable the accommodation of C(-1) in a crucial wobble-like pair at physiological pH. This design would provide a potential strategy for expanding the sequence space accessible to programmable trans-splicing ribozymes in RNA editing applications.
