title: Secondary structure confirmation and localization of Mg2+ ions in the mammalian CPEB3 ribozyme.
abstract: Most of today's knowledge of the CPEB3 ribozyme, one of the few small self-cleaving ribozymes known to occur in humans, is based on comparative studies with the hepatitis delta virus (HDV) ribozyme, which is highly similar in cleavage mechanism and probably also in structure. Here we present detailed NMR studies of the CPEB3 ribozyme in order to verify the formation of the predicted nested double pseudoknot in solution. In particular, the influence of Mg(2+), the ribozyme's crucial cofactor, on the CPEB3 structure is investigated. NMR titrations, Tb(3+)-induced cleavage, as well as stoichiometry determination by hydroxyquinoline sulfonic acid fluorescence and equilibrium dialysis, are used to evaluate the number, location, and binding mode of Mg(2+)ions. Up to eight Mg(2+)ions interact site-specifically with the ribozyme, four of which are bound with high affinity. The global fold of the CPEB3 ribozyme, encompassing 80%-90% of the predicted base pairs, is formed in the presence of monovalent ions alone. Low millimolar concentrations of Mg(2+)promote a more compact fold and lead to the formation of additional structures in the core of the ribozyme, which contains the inner small pseudoknot and the active site. Several Mg(2+)binding sites, which are important for the functional fold, appear to be located in corresponding locations in the HDV and CPEB3 ribozyme, demonstrating the particular relevance of Mg(2+)for the nested double pseudoknot structure.
authors.affiliation: Department of Chemistry, University of Zurich, CH-8057 Zurich, Switzerland.
authors.forename: Miriam
authors.initials: M
authors.lastname: Skilandat
authors.affiliation: Department of Chemistry, University of Zurich, CH-8057 Zurich, Switzerland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Rowinska-Zyrek
authors.affiliation: Department of Chemistry, University of Zurich, CH-8057 Zurich, Switzerland roland.sigel@chem.uzh.ch.
authors.forename: Roland K O
authors.initials: RK
authors.lastname: Sigel
mesh_terms: D000818:Animals; D006801:Humans; D008274:Magnesium / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D002679:Pan troglodytes; D016337:RNA, Catalytic / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry*
chemical_list: C513866:CPEB3 protein, human; D016337:RNA, Catalytic; D016601:RNA-Binding Proteins; D008274:Magnesium
keywords: CPEB3; Mg2+; NMR; metal ion; pseudoknot; ribozyme