title: Substrate selection rules for the hairpin ribozyme determined by in vitro selection, mutation, and analysis of mismatched substrates.
abstract: Substrate recognition by the hairpin ribozyme has been proposed to involve two short intermolecular helices, termed helix 1 and helix 2. We have used a combination of three methods (cleavage of mismatched substrates, in vitro selection, and site-specific mutational analysis) to systematically determine the substrate recognition rules for this RNA enzyme. Assays measuring substrate cleavage in trans under multiple turnover conditions were conducted using the wild-type ribozyme and substrates containing mismatches in all sites potentially recognized by the ribozyme. Molecules containing single- and multiple-base mismatches in helix 2 at sites distant from the cleavage site (g-4c, u-5a, g-4c: u-5a) were cleaved with reduced efficiency, whereas those with mismatches proximal to the cleavage site (c-2a, a-3c, c-2a: a-3c) were not cut. Analogous results were obtained for helix 1, where mismatches distal from the cleavage site (u+7a, u+8a, u+9a, u+7a: u+8a: u+9a) were used much more efficiently than those proximal to the cleavage site (c+4a, u-5a, g+6c, c+4a: u+5a: g+6c). In vitro selection experiments were carried out to identify active variants from populations of molecules in which either helix 1 or helix 2 was randomized. Results constitute an artificial phylogenetic data base that proves base-pairing of nucleotides at five positions within helix 1 and three positions within helix 2 and reveals a significant sequence bias at 3 bp (c+4.G6, c-2.G11, and a-3.U12). This sequence bias was confirmed at two sites by measuring relative cleavage rates of all 16 possible dinucleotide combinations at base pairs c+4.G6 and c-2.G11.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Microbiology, University of Vermont, Burlington 05405.
authors.forename: S
authors.initials: S
authors.lastname: Joseph
authors.forename: A
authors.initials: A
authors.lastname: Berzal-Herranz
authors.forename: B M
authors.initials: BM
authors.lastname: Chowrira
authors.forename: S E
authors.initials: SE
authors.lastname: Butcher
authors.forename: J M
authors.initials: JM
authors.lastname: Burke
mesh_terms: D001483:Base Sequence; D004247:DNA; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*; D012641:Selection, Genetic; D013379:Substrate Specificity
chemical_list: D016337:RNA, Catalytic; D012313:RNA; D004247:DNA