The Tetrahymena rRNA intron self-splices in E. coli: in vivo evidence for the importance of key base-paired regions of RNA for RNA enzyme function.
We have developed an in vivo RNA splicing assay for the self-splicing rRNA intron of Tetrahymena thermophila using E. coli as the host. A DNA fragment containing the intron sequence has been cloned into M13mp83 so that expression of the beta-galactosidase alpha-fragment is dependent upon intron excision from the mRNA precursor. Plaque phenotypes correlate well with levels of excised intron RNA. Point mutations were made by oligonucleotide-directed mutagenesis in conserved sequences P, Q, and S. All showed reduced splicing, agreeing with mitochondrial genetic data for S and providing the first direct evidence that P and Q are functionally important. The results support the hypothesis that base-pairing of R with S and P with Q is important for intron structure and function.
D001482:Base Composition; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D009154:Mutation; D012326:RNA Splicing; D012335:RNA, Ribosomal / Q000032:analysis*; D013768:Tetrahymena / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
D012335:RNA, Ribosomal; D001616:beta-Galactosidase