Self-splicing of a mitochondrial group I intron from the cytochrome b gene of the ascomycete Podospora anserina.
We have shown that the second intron of the Podospora mitochondrial gene coding for cytochrome b (Cytb 12) splices autocatalytically, using in vitro transcripts generated from the T7 promoter. The reaction takes place at 37 degrees C in the presence of 50 mM TRIS-HCl pH 7.5, 60 mM MgCl2 and 1 mM GTP but shows a low efficiency even at high KCl concentrations of up to 1.2 M. Under these conditions, intron bI2 follows the conventional pathway of group I splicing, and all characteristic products, with regard to both transesterification and hydrolysis, could be identified. Moreover, the intron is capable of undergoing cyclization, thereby releasing the noncoded G and one additional nucleotide (U) from the 5' end. The 5' cleavage site is preceded by the same two nucleotides, indicating a base-pairing at the same site of the internal guide sequence (IGS) for both splicing and cyclization ("one-binding-site model"). In addition, products resulting from site-specific hydrolysis 138 nucleotides downstream of the 5' splice site were detected. Unusually, the shortened intron is also able to form a circular RNA and an alternative sequence that aligns the cyclization site to the catalytic core of the intron must be assumed.
Schmidt
U
U
Technische Universität Berlin, Fachgebiet Mikrobiologie und Genetik, FRG.
Budde
E
E
Stahl
U
U
D001203:Ascomycota / Q000235:genetics*; D001483:Base Sequence; D003573:Cytochrome b Group / Q000235:genetics*; D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D004591:Electrophoresis, Polyacrylamide Gel; D006160:Guanosine Triphosphate / Q000378:metabolism; D007438:Introns; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism*; D012326:RNA Splicing; D012331:RNA, Fungal / Q000378:metabolism; D000077278:RNA, Mitochondrial; D015183:Restriction Mapping; D016415:Sequence Alignment
D003573:Cytochrome b Group; D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D012331:RNA, Fungal; D000077278:RNA, Mitochondrial; D012313:RNA; D006160:Guanosine Triphosphate