title: An RNA binding motif in the Cbp2 protein required for protein-stimulated RNA catalysis.
abstract: The fifth and terminal intron of yeast cytochrome b pre-mRNA (a group I intron) requires a protein encoded by the nuclear gene CBP2 for splicing. Because catalysis is intrinsic to the RNA, the protein is believed to promote formation of secondary and tertiary structure of the RNA, resulting in a catalytically competent intron. In vitro, this mitochondrial intron can be made to self-splice or undergo protein-facilitated splicing by varying the Mg(2+) and monovalent salt concentrations. This two-component system, therefore, provides a good model for understanding the role of proteins in RNA folding. A UV cross-linking experiment was initiated to identify RNA binding sites on Cbp2 and gain insights into Cbp2-intron interactions. A 12-amino acid region containing a presumptive contact site near the amino terminus was targeted for mutagenesis, and mutant proteins were characterized for RNA binding and stimulation of splicing. Mutations in this region resulted in partial or complete loss of function, demonstrating the importance of this determinant for stimulation of RNA splicing. Several of the mutations that severely reduced splicing did not significantly shift the overall binding isotherm of Cbp2 for the precursor RNA, suggesting that contacts critical for activity are not necessarily reflected in the dissociation constant. This analysis has identified a unique RNA binding motif of alternating basic and aromatic residues that is essential for protein facilitated splicing.
authors.affiliation: Department of Molecular Genetics, University of Florida College of Medicine, Gainesville, Florida 32605, USA.
authors.forename: H K
authors.initials: HK
authors.lastname: Tirupati
authors.forename: L C
authors.initials: LC
authors.lastname: Shaw
authors.forename: A S
authors.initials: AS
authors.lastname: Lewin
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D012331:RNA, Fungal / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012261:Ribonucleoproteins; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D014158:Transcription, Genetic
chemical_list: C071249:CBP2 protein, S cerevisiae; D003573:Cytochrome b Group; D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors; D012331:RNA, Fungal; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins