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.
Koch
J L
JL
Department of Molecular Genetics, Ohio State University, Columbus 43210.
Boulanger
S C
SC
Dib-Hajj
S D
SD
Hebbar
S K
SK
Perlman
P S
PS
D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007438:Introns; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014158:Transcription, Genetic
D004271:DNA, Fungal; D004272:DNA, Mitochondrial; D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors