Structural investigation of the effect of magnesium-ion binding on stem-loop V of the Neurospora VS ribozyme
Campbell, Dean Omar
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An essential step in the substrate recognition of the Neurospora VS ribozyme is the formation of a loop-loop interaction between the terminal loops of stem-loop I and stem-loop V. Site-specific substitution and mutagenesis have suggested that this interaction is facilitated by the presence of a U-turn in the loop of stem-loop V. We have determined the structure of stem-loop V by nuclear magnetic resonance (NMR) spectroscopy and show that it adopts a U-turn conformation, a common motif found in RNA. Structural comparisons indicate that the U-turn of stem-loop V (SL5) fulfills some but not all of the structural characteristics found in canonical U-turn motifs. The formation of the stem-loop I / stem-loop V interaction is magnesium-ion dependent. Chemical shift mapping indicated that the addition of magnesium ions to SL5 resulted in a conformational change in loop. We have determined the structure of SL5 in the presence of magnesium ions and show that the binding of magnesium ions to the loop causes a conformational change. This conformational change results in the U-turn motif of SL5 having more characteristics of a canonical U-turn motif in the presence of magnesium ions. We used paramagnetic line broadening effect of manganese ions to localize four divalent metal ion binding sites in the loop of stem-loop V. Three of these divalent metal ion binding sites are specific to the U-turn motifs and have been found in structures of other U-turn motifs. We also show that the U-turn conformation in stem-loop V allows the bases involved in the stem loop I / stem-loop V interaction (G697, A698, and C699) to be accessible, by exposing their Watson-Crick faces to the solvent. This study brings some light on the mechanism of substrate recognition in the VS ribozyme and expands our understanding of the role of U-turn motifs in RNA structure and function.