Specific Metal Ion Binding Sites in a Model of the P4-P6 Triple Helical Domain of a Group 1 Intron
Divalent metal ions are important for the tertiary folding and catalytic activity of RNA. Phosphorothioate substitution and manganese rescue experiments can be used to identify specific magnesium binding sites in the RNA structure. When Mn2+ can rescue a structure lost by sulfur substitution in the presence of Mg2+, this is interpreted to mean that the substituted oxygen is a ligand to a structurally important Mg2+ ion. Here we have performed phosphorothioate substitutions in a synthetic model system of the P4-P6 triple helical domain in the bacteriophage T4 nrdB group I intron, which has a core sequence analogy with the Tetrahymena thermophilia intron. Rp and Sp sulfur substitutions were introduced into two adjacent nucleotides positioned at the 3'-end of helix P6 (U452) and in the joining region J6/7 (U453). We have used circular dichroism (CD) spectroscopy to reveal the effects of sulfur substitution on triple helix formation in presence of either magnesium or manganese. The results show that the pro-Sp oxygens of U452 and U453 both are acting as ligands for a structurally important magnesium ion, while no such effect is seen for the pro-Rp oxygen of U452. The importance of the pro-Rp oxygen of U453 is less clear, since manganese is not able to restore the triple helical interactions within the isolated model system. However, a structural involvement of the pro-Rp oxygen of U453 for triple helix formation is emphasized.
K. Sandström, M. Lindqvist, R. Strömberg1 and A. Gräslund
Department of Biophysics, Arrhenius Laboratories