Binding of the CBP2 protein to a yeast mitochondrial group I intron requires the catalytic core of the RNA.
The yeast CBP2 gene product is required for the splicing of the terminal intron (bI5) of the mitochondrial cytochrome b pre-mRNA in vivo. In vitro, bI5 RNA self-splices efficiently only at high MgCl2 concentrations (50 mM); at 5 mM MgCl2, efficient splicing requires purified CBP2 protein. To determine the sequences within bI5 recognized by the protein, we have constructed deletion and substitution mutants of the RNA. Their binding to CBP2 was assessed by their ability to inhibit protein-dependent splicing of the wild-type bI5 RNA. Several regions, including the large L1 and L8 loops, can be deleted without affecting binding. They can therefore be eliminated from consideration as critical recognition elements. In contrast, other changes prevent the RNA from binding CBP2 and also impair self-splicing. Thus, either the catalytic core contacts the protein directly, or the integrity of the core is required for proper display of other RNA sequences that bind the protein. The results are consistent with a model in which the CBP2 protein facilitates splicing by binding to and stabilizing the active structure of the RNA. However, a more specific model is proposed in which the protein specifically enhances Mg2+ binding required for catalysis.
Gampel
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Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Cech
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D001483:Base Sequence; D002872:Chromosome Deletion; D003573:Cytochrome b Group / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005800:Genes, Fungal; D007438:Introns; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012326:RNA Splicing; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics*; D012261:Ribonucleoproteins; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
C071249:CBP2 protein, S cerevisiae; D003573:Cytochrome b Group; D005656:Fungal Proteins; D012331:RNA, Fungal; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins