title: Biochemical analysis of pistol self-cleaving ribozymes.
abstract: Pistol RNAs are members of a distinct class of self-cleaving ribozymes that was recently discovered by using a bioinformatics search strategy. Several hundred pistol ribozymes share a consensus sequence including 10 highly conserved nucleotides and many other modestly conserved nucleotides associated with specific secondary structure features, including three base-paired stems and a pseudoknot. A representative pistol ribozyme from the bacterium Lysinibacillus sphaericus was found to promote RNA strand scission with a rate constant of ∼10 min(-1) under physiological Mg(2+) and pH conditions. The reaction proceeds via the nucleophilic attack of a 2'-oxygen atom on the adjacent phosphorus center, and thus adheres to the same general catalytic mechanism of internal phosphoester transfer as found with all other classes of natural self-cleaving ribozymes discovered to date. Analyses of the kinetic characteristics and the metal ion requirements of the cleavage reaction reveal that members of this ribozyme class likely use several catalytic strategies to promote the rapid cleavage of RNA.
authors.affiliation: Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520-8103, USA Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA.
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Harris
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA.
authors.forename: Christina E
authors.initials: CE
authors.lastname: Lünse
authors.affiliation: Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520-8103, USA Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA.
authors.forename: Sanshu
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8103, USA.
authors.forename: Kenneth I
authors.initials: KI
authors.lastname: Brewer
authors.affiliation: Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520-8103, USA Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8103, USA.
authors.forename: Ronald R
authors.initials: RR
authors.lastname: Breaker
mesh_terms: D001419:Bacteria / Q000235:genetics; D001483:Base Sequence; D002384:Catalysis; D019295:Computational Biology / Q000379:methods; D016384:Consensus Sequence / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000235:genetics; D012313:RNA / Q000235:genetics*; D016337:RNA, Catalytic / Q000235:genetics*
chemical_list: D009711:Nucleotides; D016337:RNA, Catalytic; D012313:RNA
keywords: RNA cleavage; RNA processing; comparative sequence analysis; phosphoester transfer; phosphorothioate