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 domain...

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Bibliographic Details
Published in:Biochemistry (Easton) 2000-03, Vol.39 (9), p.2174-2182
Main Authors: Butcher, Samuel E, Allain, Frédéric H.-T, Feigon, Juli
Format: Article
Language:English
Subjects:
Binding Sites
Cations, Divalent
Cations, Monovalent
Manganese - chemistry
Manganese - metabolism
Metals - chemistry
Metals - metabolism
Models, Molecular
Nuclear Magnetic Resonance, Biomolecular
Nucleic Acid Conformation
RNA, Catalytic - chemistry
RNA, Catalytic - metabolism
Solutions
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Summary: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 Mn2+, Mg2+, and Co(NH3)6 3+. There is no significant structural change in the B domain upon the addition of Co(NH3)6 3+ or Mg2+. No specific monovalent ion binding sites exist on the B domain, as determined by 15NH4 + 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 Mn2+ 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(H2O)6 2+ or Co(NH3)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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi9923454