title: Intermolecular domain docking in the hairpin ribozyme: metal dependence, binding kinetics and catalysis.
abstract: The hairpin ribozyme is a prototype small, self-cleaving RNA motif. It exists naturally as a four-way RNA junction containing two internal loops on adjoining arms. These two loops interact in a cation-driven docking step prior to chemical catalysis to form a tightly integrated structure, with dramatic changes occurring in the conformation of each loop upon docking. We investigate the thermodynamics and kinetics of the docking process using constructs in which loop A and loop B reside on separate molecules. Using a novel CD difference assay to isolate the effects of metal ions linked to domain docking, we find the intermolecular docking process to be driven by sub-millimolar concentrations of the exchange-inert Co(NH 3) 6 (3+). RNA self-cleavage requires binding of lower-affinity ions with greater apparent cooperativity than the docking process itself, implying that, even in the absence of direct coordination to RNA, metal ions play a catalytic role in hairpin ribozyme function beyond simply driving loop-loop docking. Surface plasmon resonance assays reveal remarkably slow molecular association, given the relatively tight loop-loop interaction. This observation is consistent with a "double conformational capture" model in which only collisions between loop A and loop B molecules that are simultaneously in minor, docking-competent conformations are productive for binding.
authors: Department of Biochemistry and Molecular Biology; Michigan State University; East Lansing, MI USA.
authors: Minako
authors: M
authors: Sumita
authors: Neil A
authors: NA
authors: White
authors: Kristine R
authors: KR
authors: Julien
authors: Charles G
authors: CG
authors: Hoogstraten
mesh_terms: D001665:Binding Sites; D055162:Biocatalysis; D002942:Circular Dichroism; D003035:Cobalt / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
chemical_list: D016337:RNA, Catalytic; C418481:hairpin ribozyme; C030862:cobalt ammonium complex; D003035:Cobalt
keywords: RNA catalysis; RNA-cation interactions; circular dichroism; hairpin ribozyme; ribozyme kinetics; surface plasmon resonance