title: Essential roles of innersphere metal ions for the formation of the glutamine binding site in a bifunctional ribozyme.
abstract: Numerous studies on naturally occurring ribozymes have shown that the functional roles of metal ions in promoting RNA catalysis are diverse. Earlier studies performed on the in vitro selected aminoacyl-transferase ribozyme (ATRib) have revealed that a fully hydrated Mg2+ ion plays an essential role in catalysis [Suga, H., Cowan, J. A., and Szostak, J. W. (1998) Biochemistry 28, 10118-10125]. More recently, we have evolved this ATRib into a bifunctional ribozyme, called AD02 [Lee, N., et al. (2000) Nat. Struct. Biol. 7, 28-33]. This new ribozyme consists of two catalytic domains, the original ATRib domain and a new glutamine-recognition (QR) domain, and exhibits a function of charging glutamine to tRNA. Here we elucidate crucial roles of metal ions involved in the QR domain, that are distinct from those in the ATRib domain. The metal ions in the QR domain require innersphere coordinations, and both Mg2+ and Ca2+ can support catalysis. Extensive Tb3+-Mg2+ and Tb3+-Co(NH3)6(3+) competition cleavage experiments have shown that the QR domain has high and low affinity metal binding sites, which are involved in the Mg2+-dependent structural alteration to form the glutamine binding site [Lee, N., and Suga, H. (2001) RNA 7, 1043-1051]. Kinetic studies in the presence of divalent and monovalent ions have suggested that the essential role of the metal ions in the QR domain is most likely structural.
authors.affiliation: Department of Chemistry, 657 Natural Sciences Complex, University at Buffalo, State University of New York, Buffalo, New York 14260-3000, USA.
authors.forename: N
authors.initials: N
authors.lastname: Lee
authors.forename: H
authors.initials: H
authors.lastname: Suga
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003035:Cobalt / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005973:Glutamine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D016337:RNA, Catalytic / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008670:Metals; D016337:RNA, Catalytic; D005973:Glutamine; C030862:cobalt ammonium complex; D003035:Cobalt; D008274:Magnesium