SELF-ASSEMBLY OF A GROUP-I INTRON ACTIVE-SITE FROM ITS COMPONENT TERTIARY STRUCTURAL DOMAINS

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 h elical segments and conserved unpaired nucleotides, and the isolated P 4-P6 domain has been shown to have substantial native tertiary structu re. The proposed tertiary structure domains of the Tetrahymena intron were synthesized separately and shown to self-assemble into a catalyti cally active complex. Surprisingly, the concentration dependence of th ese reactions revealed that the domains interact with nanomolar appare nt dissociation constants, even though there is no known base pairing between P4-P6 and P3-P9. This suggests that the domains interact throu gh 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 demonstrate d with the permuted molecule when nucleotides proposed to form a tripl e-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 orie ntation of the RNA domains or participate more directly in catalysis.