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

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.

Group II introns deleted for multiple substructures retain self-splicing activity.

Group II introns can be folded into highly conserved secondary structures with six major substructures or domains. Domains 1 and 5 are known to play key roles in self-splicing, while the roles of domains 2, 3, 4, and 6 are less clear. A trans assay for domain 5 function has been developed which indicates that domain 5 has a binding site on the precursor RNA that is not predicted from any secondary structure element. In this study, the self-splicing group II intron 5 gamma of the coxI gene of yeast mitochondrial DNA was deleted for various intron domains, singly and in combinations. Those mutant introns were characterized for self-splicing reactions in vitro as a means of locating the domain 5 binding site. A single deletion of domain 2, 3, 4, or 6 does not block in vitro reactions at either splice junction, though the deletion of domain 6 reduces the fidelity of 3' splice site selection somewhat. Even the triple deletion lacking domains 2, 4, and 6 retains some self-splicing activity. The deletion of domains 2, 3, 4, and 6 blocks the reaction at the 3' splice junction but not at the 5' junction. From these results, we conclude that the binding site for domain 5 is within domain 1 and that the complex of 5' exon, domain 1, and domain 5 (plus short connecting sequences) constitutes the essential catalytic core of this intron.

A maturase-encoding group IIA intron of yeast mitochondria self-splices in vitro.

Intron 1 of the coxI gene of yeast mitochondrial DNA (aI1) is a group IIA intron that encodes a maturase function required for its splicing in vivo. It is shown here to self-splice in vitro under some reaction conditions reported earlier to yield efficient self-splicing of group IIB introns of yeast mtDNA that do not encode maturase functions. Unlike the group IIB introns, aI1 is inactive in 10 mM Mg2+ (including spermidine) and requires much higher levels of Mg2+ and added salts (1M NH4Cl or KCl or 2M (NH4)2SO4) for ready detection of splicing activity. In KCl-stimulated reactions, splicing occurs with little normal branch formation; a post-splicing reaction of linear excised intron RNA that forms shorter lariat RNAs with branches at cryptic sites was evident in those samples. At low levels of added NH4Cl or KCl, the precursor RNA carries out the first reaction step but appears blocked in the splicing step. AI1 RNA is most reactive at 37-42 degrees C, as compared with 45 degrees C for the group IIB introns; and it lacks the KCl- or NH4Cl-dependent spliced-exon reopening reaction that is evident for the self-splicing group IIB introns of yeast mitochondria. Like the group IIB intron aI5 gamma, the domain 4 of aI1 can be largely deleted in cis, without blocking splicing; also, trans-splicing of half molecules interrupted in domain 4 occurs. This is the first report of a maturase-encoding intron of either group I or group II that self-splices in vitro.