Domain 5 interacts with domain 6 and influences the second transesterification reaction of group II intron self-splicing

Abstract

The role of domain 5 (d5) from the self-splicing group II intron 5 gamma of the COXI gene of yeast mitochondrial DNA in branching and 3' splice site utilization has been studied using a substrate transcript lacking d5 (delta d5 RNA). This RNA is completely unreactive in vitro, but releases 5' exon by hydrolysis under various reaction conditions when d5 RNA is added in trans. Under an extreme reaction condition, some accurate branching and splicing occur. Much more efficient use of a 3' splice site is obtained when delta d5 RNA is complemented by a transcript containing the wild-type domains 5 and 6 plus the 3' exon. While most delta d5 RNA molecules in that protocol still react by hydrolysis at the 5' splice site, the branching that occurs uses only the d6 tethered to d5 that is provided in trans. The use of this d6 and the 3' splice site also linked to d5, along with the observed indifference to the other d6 and 3' splice site resident in the delta d5 RNA, indicates that d5 plays a key role in positioning d6 for the first reaction step as well as in 3' splice site use. Two models for the manner by which d5 interacts with d6 are discussed.