Cotranscriptional splicing of a group I intron is facilitated by the Cbp2 protein.
The nuclear CBP2 gene encodes a protein essential for the splicing of a mitochondrial group I intron in Saccharomyces cerevisiae. This intron (bI5) is spliced autocatalytically in the presence of high concentrations of magnesium and monovalent salt but requires the Cbp2 protein for splicing under physiological conditions. Addition of Cbp2 during RNA synthesis permitted cotranscriptional splicing. Splicing did not occur in the transcription buffer in the absence of synthesis. The Cbp2 protein appeared to modify the folding of the intron during RNA synthesis: pause sites for RNA polymerase were altered in the presence of the protein, and some mutant transcripts that did not splice after transcription did so during transcription in the presence of Cbp2. Cotranscriptional splicing also reduced hydrolysis at the 3' splice junction. These results suggest that Cbp2 modulates the sequential folding of the ribozyme during its synthesis. In addition, splicing during transcription led to an increase in RNA synthesis with both T7 RNA polymerase and mitochondrial RNA polymerase, implying a functional coupling between transcription and splicing.
Lewin
A S
AS
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville 32610-0266, USA.
Thomas
J
J
Tirupati
H K
HK
D001483:Base Sequence; D004272:DNA, Mitochondrial / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005091:Exons; D005656:Fungal Proteins / Q000378:metabolism*; D017403:In Situ Hybridization; D007438:Introns; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D012326:RNA Splicing; D012331:RNA, Fungal / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D000077278:RNA, Mitochondrial; D012261:Ribonucleoproteins; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014158:Transcription, Genetic; D014764:Viral Proteins
C071249:CBP2 protein, S cerevisiae; D004272:DNA, Mitochondrial; D005656:Fungal Proteins; D012331:RNA, Fungal; D000077278:RNA, Mitochondrial; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases