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Nucleic Acids Research, 1993, Vol. 21, No. 8 1797-1804
© 1993

MOLECULAR BIOLOGY

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

Sulayman D. Dib-Hajj+, Scott C. Boulanger1, Sharda K. Hebbar, Craig L. Peebles2, James S. Franzen2 and Philip S. Perlman1,*

1MCDB Program and Department of Molecular Genetics, The Ohio State University 484 W. 12th Avenue, Columbus, OH 43210, 1Department of Biochemistry, University of Texas Southwestern Medical Center 5323 Harry Hines Blvd, Dallas, TX 75235–9038 2Department of Biological Sciences, University of Pittsburgh Pittsburgh, PA 15260, USA

* To whom correspondence should be addressed

Received January 25, 1993. Revised March 18, 1993. Accepted March 18, 1993.

The role of domain 5 (d5) from the self-splicing group II Intron 5{gamma} of the COXI gene of yeast mitochondrlal 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.

+ Present address: Department of Entomology, Texas A&M University, College Station, TX 77843, USA


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