Determination of metal ion binding sites within the hairpin ribozyme domains by NMR.
Cations play an important role in RNA folding and stabilization. The hairpin ribozyme is a small catalytic RNA consisting of two domains, A and B, which interact in the transition state in an ion-dependent fashion. Here we describe the interaction of mono-, di-, and trivalent cations with the domains of the ribozyme, as studied by homo- and heteronuclear NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping, and intermolecular NOEs indicate that the B domain contains four to five metal binding sites, which bind Mn(2+), Mg(2+), and Co(NH(3))(6)(3+). There is no significant structural change in the B domain upon the addition of Co(NH(3))(6)(3+) or Mg(2+). No specific monovalent ion binding sites exist on the B domain, as determined by (15)NH(4)(+) binding studies. In contrast to the B domain, there are no observable metal ion interactions within the internal loop of the A domain. Model structure calculations of Mn(2+) interactions at two sites within the B domain indicate that the binding sites comprise major groove pockets lined with functional groups oriented so that multiple hydrogen bonds can be formed between the RNA and Mn(H(2)O)(6)(2+) or Co(NH(3))(6)(3+). Site 1 is very similar in geometry to a site within the P4-P6 domain of the Tetrahymena group I intron, while site 2 is unique among known ion binding sites. The site 2 ion interacts with a catalytically essential nucleotide and bridges two phosphates. Due to its location and geometry, this ion may play an important role in the docking of the A and B domains.
Butcher
S E
SE
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-1569, USA.
Allain
F H
FH
Feigon
J
J
D001665:Binding Sites; D002413:Cations, Divalent; D002414:Cations, Monovalent; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D012996:Solutions
D002413:Cations, Divalent; D002414:Cations, Monovalent; D008670:Metals; D016337:RNA, Catalytic; D012996:Solutions; D008345:Manganese