title: Identification of the catalytic subdomain of the VS ribozyme and evidence for remarkable sequence tolerance in the active site loop.
abstract: We show here that the ribozyme domain of the Neurospora VS ribozyme consists of separable upper and lower subdomains. Deletion analysis demonstrates that the entire upper subdomain (helices III/IV/V) is dispensable for site-specific cleavage activity, providing experimental evidence that the active site is contained within the lower subdomain and within the substrate itself. We demonstrate an important role in cleavage activity for a region of helix VI called the 730 loop. Surprisingly, several loop sequences, sizes, and structures at this position can support site-specific cleavage, suggesting that a variety of non-Watson-Crick structures, rather than a specific loop structure, in this region of the ribozyme can contribute to formation of the active site.
authors.affiliation: Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada M5S 1A8, USA.
authors.forename: Vanita D
authors.initials: VD
authors.lastname: Sood
authors.forename: Richard A
authors.initials: RA
authors.lastname: Collins
mesh_terms: D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009491:Neurospora / Q000201:enzymology* / Q000235:genetics*; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012331:RNA, Fungal / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity
chemical_list: D016337:RNA, Catalytic; D012331:RNA, Fungal; D004722:Endoribonucleases; C438195:varkud satellite ribozyme