The phylogenetically predicted base-pairing interaction between alpha and alpha' is required for group II splicing in vitro

Abstract

The correct folding of group II introns apparently depends on multiple tertiary base-pairing interactions. Understanding the relationship between spliceosome and group II splicing systems ultimately requires a three-dimensional model for both structures. In turn, successful modeling depends at least in part on identifying tertiary base pairings. Sequence elements alpha and alpha' are partners in a potential interaction of approximately 6 base pairs that can be identified within domain 1 of most group II introns. In comparisons between related introns, alpha and alpha' maintain their potential for Watson-Crick base pairing, even though their primary sequences can vary [Michel, F., Umesono, K. & Ozeki, H. (1989) Gene 82, 5-30]. Substitutions were constructed at alpha and alpha' for a block of 6 bases each in the group II intron a5 gamma, the last intron of the COXI gene from the mitochondrial DNA of Saccharomyces cerevisiae. Each substitution was defective for self-splicing, while the compensatory double derivative was restored to active splicing. The alpha-alpha' interaction is required for the first step of splicing--that is, recognition of the 5' splice junction and transesterification with the branch site--since the derivative transcripts displayed little or no activity. The compensatory double derivative produced lariat introns and spliced exons with normal structures, showing that splicing activity and precise recognition were restored. We conclude that the alpha-alpha' base pairing is necessary for efficient self-splicing by intron a5 gamma under several conditions. This result also provides an additional constraint for any three-dimensional model of group II intron structure.