title: Probing non-selective cation binding in the hairpin ribozyme with Tb(III).
abstract: Catalysis by the hairpin ribozyme is stimulated by a wide range of both simple and complex metallic and organic cations. This independence from divalent metal ion binding unequivocally excludes inner-sphere coordination to RNA as an obligatory role for metal ions in catalysis. Hence, the hairpin ribozyme is a unique model to study the role of outer-sphere coordinated cations in folding of a catalytically functional RNA structure. Here, we demonstrate that micromolar concentrations of a deprotonated aqueous complex of the lanthanide metal ion terbium(III), Tb(OH)(aq)(2+), reversibly inhibit the ribozyme by competing for a crucial, yet non-selective cation binding site. Tb(OH)(aq)(2+) also reports a likely location of this binding site through backbone hydrolysis, and permits the analysis of metal binding through sensitized luminescence. We propose that the critical cation-binding site is located at a position within the catalytic core that displays an appropriately-sized pocket and a high negative charge density. We show that cationic occupancy of this site is required for tertiary folding and catalysis, yet the site can be productively occupied by a wide variety of cations. It is striking that micromolar Tb(OH)(aq)(2+) concentrations are compatible with tertiary folding, yet interfere with catalysis. The motif implicated here in cation-binding has also been found to organize the structure of multi-helix loops in evolutionary ancient ribosomal RNAs. Our findings, therefore, illuminate general principles of non-selective outer-sphere cation binding in RNA structure and function that may have prevailed in primitive ribozymes of an early "RNA world".
authors.affiliation: Markey Center for Molecular Genetics, Department of Microbiology and Molecular Genetics, The University of Vermont, 306 Stafford Hall, Burlington, VT, 05405, USA. nwalter@umich.edu
authors.forename: N G
authors.initials: NG
authors.lastname: Walter
authors.forename: N
authors.initials: N
authors.lastname: Yang
authors.forename: J M
authors.initials: JM
authors.lastname: Burke
mesh_terms: D001483:Base Sequence; D001667:Binding, Competitive / Q000187:drug effects; D002384:Catalysis / Q000187:drug effects; D002412:Cations / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation / Q000187:drug effects; D011522:Protons; D016337:RNA, Catalytic / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013725:Terbium / Q000378:metabolism* / Q000494:pharmacology; D013816:Thermodynamics
chemical_list: D002412:Cations; D011522:Protons; D016337:RNA, Catalytic; D013725:Terbium