Nuclear Magnetic Resonance Structure of the Varkud Satellite Ribozyme Stem−Loop V RNA and Magnesium-Ion Binding from Chemical-Shift Mapping^,

Abstract

An important step in the substrate recognition of the Neurospora Varkud Satellite (VS) ribozyme is the formation of a magnesium-dependent loop/loop interaction between the terminal loops of stem-loops I and V. We have studied the structure of stem-loop V by nuclear magnetic resonance spectroscopy and shown that it adopts a U-turn conformation, a common motif found in RNA. Structural comparisons indicate that the U-turn of stem-loop V fulfills some but not all of the structural characteristics found in canonical U-turn structures. This U-turn conformation exposes the Watson-Crick faces of the bases within stem-loop V (G697, A698, and C699) and makes them accessible for interaction with stem-loop I. Using chemical-shift mapping, we show that magnesium ions interact with the loop of the isolated stem-loop V and induce a conformational change that may be important for interaction with stem-loop I. This study expands our understanding of the role of U-turn motifs in RNA structure and function and provides insights into the mechanism of substrate recognition in the VS ribozyme.

Role of SLV in SLI substrate recognition by the Neurospora VS ribozyme

“…Although Mg 2+ ions affect the loop conformation, they do not significantly change the conformation of the three SLV bases (G697, A698, and C699) that are proposed to base pair with SLI (Rastogi et al 1996). In both structures, these three bases of SLV are exposed and well positioned for binding to SLI (Campbell and Legault 2005;Campbell et al 2006). The most significant change induced by Mg 2+ ions occurs at the extruded loop residue U700, which comes closer to the three interacting bases (G697, A698, and C699) of SLV ( Fig.…”

“…FIGURE 2. NMR structures of the SLV loop determined in the absence (left) and presence (right) of magnesium ions (Campbell and Legault 2005;Campbell et al 2006), showing the large conformational change of U700 (blue). The dotted lines represent two hydrogen bonds that are characteristic of canonical U-turn structures, the A698 N7 to U696 29OH (blue) and the U696 H3 to A698 39-phosphate interactions (red).…”

“…1A; Beattie et al 1995), tertiary structure models (Hiley and Collins 2001;Lafontaine et al 2002), and NMR structures of individual stem-loops (Michiels et al 2000;Dieckmann 2001, 2004;Hoffmann et al 2003;Campbell and Legault 2005;Campbell et al 2006). The catalytic domain of the VS ribozyme consists of five stem-loop subdomains (SLII-SLVI) (Fig.…”

“…2), one in the absence (SLV free ) (Campbell and Legault 2005) and one in the presence of magnesium ions (SLV Mg ) (Campbell et al 2006). The loop of SLV free forms a loose noncanonical U-turn motif, whereas that of SLV Mg forms a compact canonical U-turn motif (Campbell and Legault 2005;Campbell et al 2006). The U-turn of SLV free was termed noncanonical, because it lacks the stacking interaction between the U696 base and the A698 59-phosphate and the hydrogen bond between the U696 H3 and the A698 39-phosphate (Fig.…”

“…The U-turn of SLV free was termed noncanonical, because it lacks the stacking interaction between the U696 base and the A698 59-phosphate and the hydrogen bond between the U696 H3 and the A698 39-phosphate (Fig. 2) that are characteristic of canonical U-turn structures and found in SLV Mg (Campbell and Legault 2005;Campbell et al 2006). Although Mg 2+ ions affect the loop conformation, they do not significantly change the conformation of the three SLV bases (G697, A698, and C699) that are proposed to base pair with SLI (Rastogi et al 1996).…”