title: Cobalt(III)hexaammine-dependent photocrosslinks in the hairpin ribozyme.
abstract: We have utilized the hairpin ribozyme, an RNA enzyme whose structure has been solved by high-resolution methods, to develop a new tool for mapping nucleobase-stacking interactions and potential metal-binding sites in RNA molecules. This tool involves the photoactivation of a specifically bound cobalt(III)hexaammine molecule at wavelengths corresponding to excitation of the metal ion complex only; no base excitation is involved. The photoexcitation initiates a process which strongly promotes the formation of a novel covalent bond or crosslink between one base (termed the "first base"), which is close in space to the excited cobalt(III)hexaammine complex, and another base upon which the first base is closely stacked. These crosslinked species can be isolated and sequenced; their activities can be analyzed to ensure that the crosslinked structures represent an active conformation of the molecule. We have shown that, as in electron transfer in DNA, several criteria must be met to result in the successful formation of these crosslinks. These include the appropriate oxidation potential of the first donor base, the stacking and close interaction of the two donor bases involved in the crosslink, and the binding of a specific cobalt(III)hexaammine molecule to the first donor base. Additionally, we have determined that this crosslinking is pH-sensitive, although the cause of this sensitivity remains unknown. This tool has proven useful in the past for the analysis of the hairpin ribozyme folded structure, and has been applied to identify potential metal-binding sites on the hairpin and extended hammerhead ribozymes.
authors.affiliation: Department of Microbiology and Molecular Genetics, 95 Carrigan Drive, University of Vermont, Burlington, VT 05405, USA. Electronic address: cchant@smcvt.edu.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Kraemer-Chant
authors.affiliation: Department of Microbiology and Molecular Genetics, 95 Carrigan Drive, University of Vermont, Burlington, VT 05405, USA.
authors.forename: Joyce E
authors.initials: JE
authors.lastname: Heckman
authors.affiliation: Department of Microbiology and Molecular Genetics, 95 Carrigan Drive, University of Vermont, Burlington, VT 05405, USA.
authors.forename: Dominic
authors.initials: D
authors.lastname: Lambert
authors.affiliation: Department of Microbiology and Molecular Genetics, 95 Carrigan Drive, University of Vermont, Burlington, VT 05405, USA.
authors.forename: John M
authors.initials: JM
authors.lastname: Burke
mesh_terms: D000588:Amines / Q000737:chemistry*; D003035:Cobalt / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D009942:Organometallic Compounds / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry*
chemical_list: D000588:Amines; D003432:Cross-Linking Reagents; D009942:Organometallic Compounds; D016337:RNA, Catalytic; C418481:hairpin ribozyme; D003035:Cobalt
keywords: Cobalt(III)hexaammine; Hairpin ribozyme; Photocrosslinking