Self-assembly of a group I intron active site from its component tertiary structural domains.

Abstract

The catalytic core of Group I self-splicing introns has been proposed to consist of two structural domains, P4-P6 and P3-P9. Each contains helical segments and conserved unpaired nucleotides, and the isolated P4-P6 domain has been shown to have substantial native tertiary structure. The proposed tertiary structure domains of the Tetrahymena intron were synthesized separately and shown to self-assemble into a catalytically active complex. Surprisingly, the concentration dependence of these reactions revealed that the domains interact with nanomolar apparent dissociation constants, even though there is no known base pairing between P4-P6 and P3-P9. This suggests that the domains interact through multiple tertiary contacts, the nature of which can now be explored in this system. For example, a circularly permuted version of the P4-P6 domain, which folds similarly to the native P4-P6 molecule, formed a stable but inactive complex. Interestingly, activity was demonstrated with the permuted molecule when nucleotides proposed to form a triple-strand interaction with P4 and P6 were restored as part of the P1-P3 substrate or as part of the P3-P9 RNA. Thus, beyond stabilization of the P4-P6 domain, the triple-strand region may facilitate correct orientation of the RNA domains or participate more directly in catalysis.