NMR Structure of Varkud Satellite Ribozyme Stem−Loop V in the Presence of Magnesium Ions and Localization of Metal-Binding Sites^†,‡

In the Neurospora VS ribozyme, magnesium ions facilitate formation of a loop−loop interaction between stem−loops I and V, which is important for recognition and activation of the stem−loop I substrate. Here, we present the high-resolution NMR structure of stem−loop V (SL5) in the presence of Mg²⁺ (SL5^Mg) and demonstrate that Mg²⁺ induces a conformational change in which the SL5 loop adopts a compact structure with most characteristics of canonical U-turn structures. Divalent cation-binding sites were probed with Mn²⁺-induced paramagnetic line broadening and intermolecular NOEs to Co(NH₃)₆³⁺. Structural modeling of Mn(H₂O)₆²⁺ in SL5^Mg revealed four divalent cation-binding sites in the loop. Sites 1, 3, and 4 are located in the major groove near multiple phosphate groups, whereas site 2 is adjacent to N7 of G697 and N7 of A698 in the minor groove. Cation-binding sites equivalent to sites 1−3 in SL5 are present in other U-turn motifs, and these metal-binding sites may represent a common feature of the U-turn fold. Although magnesium ions affect the loop conformation, they do not significantly change the conformation of residues 697−699 involved in the proposed Watson−Crick base pairs with stem−loop I. In both the presence and the absence of Mg²⁺, G697, A698, and C699 adopt an A-form structure that exposes their Watson−Crick faces, and this is compatible with their proposed interaction with stem−loop I. In SL5^Mg, however, U700 becomes exposed on the minor groove face of the loop in the proximity of the bases of G697, A698, and C699, suggesting that the Mg²⁺-bound conformation of stem−loop V allows additional contacts with stem−loop I. These studies improve our understanding of the role of Mg²⁺ in U-turn structures and in substrate recognition by the VS ribozyme.