title: Mutational inhibition of ligation in the hairpin ribozyme: substitutions of conserved nucleobases A9 and A10 destabilize tertiary structure and selectively promote cleavage.
abstract: The hairpin ribozyme acts as a reversible, site-specific endoribonuclease that ligates much more rapidly than it cleaves cognate substrate. While the reaction pathway for ligation is the reversal of cleavage, little is known about the atomic and electrostatic details of the two processes. Here, we report the functional consequences of molecular substitutions of A9 and A10, two highly conserved nucleobases located adjacent to the hairpin ribozyme active site, using G, C, U, 2-aminopurine, 2,6-diaminopurine, purine, and inosine. Cleavage and ligation kinetics were analyzed, tertiary folding was monitored by hydroxyl radical footprinting, and interdomain docking was studied by native gel electrophoresis. We determined that nucleobase substitutions that exhibit significant levels of interference with tertiary folding and interdomain docking have relatively large inhibitory effects on ligation rates while showing little inhibition of cleavage. Indeed, one variant, A10G, showed a fivefold enhancement of cleavage rate and no detectable ligation, and we suggest that this property may be uniquely well suited to intracellular targeted RNA cleavage applications. Results support a model in which formation of a kinetically stable tertiary structure is essential for ligation of the hairpin ribozyme, but is not necessary for cleavage.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405, USA.
authors.forename: Snigdha
authors.initials: S
authors.lastname: Gaur
authors.forename: Joyce E
authors.initials: JE
authors.lastname: Heckman
authors.forename: John M
authors.initials: JM
authors.lastname: Burke
mesh_terms: D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007700:Kinetics; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity
chemical_list: D016337:RNA, Catalytic