Specific metal-ion binding P4-P6 triple-helical domain sites in a model ofthe of a group I intron

Divalent metal ions play a crucial role in RNA structure and catalysis. Pho sphorothioate substitution and manganese rescue experiments can reveal phos phate oxygens interacting specifically with magnesium ions essential for st ructure and/or activity. In this study, phosphorothioate interference exper iments in combination with structural sensitive circular dichroism spectros copy have been used to probe molecular interactions underlying an important RNA structural motif. We have studied a synthetic model of the P4-P6 tripl e-helical domain in the bacteriophage T4 nrdB group I intron, which has a c ore sequence analogous to the Tetrahymena ribozyme. Rp and Sp sulfur substi tutions were introduced into two adjacent nucleotides positioned at the 3' end of helix P6 (U452) and in the joining region J6/7 (U453). The effects o f sulfur substitution on triple helix formation in the presence of differen t ratios of magnesium and manganese were studied by the use of difference c ircular dichroism spectroscopy. The results show that the pro-Sp oxygen of U452 acts as a ligand for a structurally important magnesium ion, whereas n o such effect is seen for the pro-Rp oxygen of U452. The importance of the pro-Rp and pro-Sp oxygens of U453 is less clear, because addition of mangan ese could not significantly restore the triple-helical interactions within the isolated substituted model systems. The interpretation is that U453 is so sensitive to structural disturbance that any change at this position hin ders the proper formation of the triple helix.