title: Tertiary structure formation in the hairpin ribozyme monitored by fluorescence resonance energy transfer.
abstract: The complex formed by the hairpin ribozyme and its substrate consists of two independently folding domains which interact to form a catalytic structure. Fluorescence resonance energy transfer methods permit us to study reversible transitions of the complex between open and closed forms. Results indicate that docking of the domains is required for both the cleavage and ligation reactions. Docking is rate-limiting for ligation (2 min-1) but not for cleavage, where docking (0.5 min-1) precedes a rate-limiting conformational transition or slow-reaction chemistry. Strikingly, most modifications to the RNA (such as a G+1A mutation in the substrate) or reaction conditions (such as omission of divalent metal ion cofactors) which inhibit catalysis do so by preventing docking. This demonstrates directly that mutations and modifications which inhibit a step following substrate binding are not necessarily involved in catalysis. An improved kinetic description of the catalytic cycle is derived, including specific structural transitions.
authors.affiliation: Markey Center for Molecular Genetics, Department of Microbiology and Molecular Genetics, The University of Vermont, Burlington, VT 05405, USA.
authors.forename: N G
authors.initials: NG
authors.lastname: Walter
authors.forename: K J
authors.initials: KJ
authors.lastname: Hampel
authors.forename: K M
authors.initials: KM
authors.lastname: Brown
authors.forename: J M
authors.initials: JM
authors.lastname: Burke
mesh_terms: D001482:Base Composition; D001665:Binding Sites; D006147:Guanine; D006863:Hydrogen-Ion Concentration; D008274:Magnesium; D008670:Metals; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D008670:Metals; D016337:RNA, Catalytic; D006147:Guanine; D008274:Magnesium