Determination of Metal Ion Binding Sites within the Hairpin Ribozyme Domains by NMR

详细信息    查看全文

• 作者：Samuel E. Butcher ; Fré ; dé ; ric H.-T. Allain ; and Juli Feigon
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：March 7, 2000
• 年：2000
• 卷：39
• 期：9
• 页码：2174 - 2182
• 全文大小：423K
• 年卷期：v.39,no.9(March 7, 2000)
• ISSN：1520-4995

文摘

Cations play an important role in RNA folding and stabilization. The hairpin ribozyme is asmall catalytic RNA consisting of two domains, A and B, which interact in the transition state in anion-dependent fashion. Here we describe the interaction of mono-, di-, and trivalent cations with thedomains of the ribozyme, as studied by homo- and heteronuclear NMR spectroscopy. Paramagnetic linebroadening, chemical shift mapping, and intermolecular NOEs indicate that the B domain contains fourto five metal binding sites, which bind Mn²⁺, Mg²⁺, and Co(NH₃)₆³⁺. There is no significant structuralchange in the B domain upon the addition of Co(NH₃)₆³⁺ or Mg²⁺. No specific monovalent ion bindingsites exist on the B domain, as determined by ¹⁵NH₄⁺ binding studies. In contrast to the B domain, thereare no observable metal ion interactions within the internal loop of the A domain. Model structurecalculations of Mn²⁺ interactions at two sites within the B domain indicate that the binding sites comprisemajor groove pockets lined with functional groups oriented so that multiple hydrogen bonds can be formedbetween the RNA and Mn(H₂O)₆²⁺ or Co(NH₃)₆³⁺. Site 1 is very similar in geometry to a site within theP4-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 itslocation and geometry, this ion may play an important role in the docking of the A and B domains.