NMR structure of the active conformation of the Varkud satellite ribozyme cleavage site

Abstract

Substrate cleavage by the Neurospora Varkud satellite (VS) ribozyme involves a structural change in the stem-loop I substrate from an inactive to an active conformation. We have determined the NMR solution structure of a mutant stem-loop I that mimics the active conformation of the cleavage site internal loop. This structure shares many similarities, but also significant differences, with the previously determined structures of the inactive internal loop. The active internal loop displays different base-pairing interactions and forms a novel RNA fold composed exclusively of sheared G-A base pairs. From chemical-shift mapping we identified two Mg2+ binding sites in the active internal loop. One of the Mg2+ binding sites forms in the active but not the inactive conformation of the internal loop and is likely important for catalysis. Using the structure comparison program mc-search, we identified the active internal loop fold in other RNA structures. In Thermus thermophilus 16S rRNA, this RNA fold is directly involved in a long-range tertiary interaction. An analogous tertiary interaction may form between the active internal loop of the substrate and the catalytic domain of the VS ribozyme. The combination of NMR and bioinformatic approaches presented here has identified a novel RNA fold and provides insights into the structural basis of catalytic function in the Neurospora VS ribozyme.

Fig. 1.

(a) Sequence and secondary structure of the Neurospora VS ribozyme. The interaction between stem-loops I and V is indicated by a dashed line, and residues involved in this interaction are circled (7). Upon tertiary folding of the ribozyme, stem-loop I (subdivided into Ia and Ib) undergoes a structural change from an inactive to an active conformation (11). The minimal substrate domain for the trans cleavage reaction is boxed (10), and the cleavage site is indicated by the arrowhead. (b) Sequence and secondary structure of SL1′ RNA. WT and mutant nucleotides are represented by uppercase and lowercase letters, respectively. (c) SL1′ is cleaved by the VS ribozyme. 3′-³²P-end-labeled SL1′ (25 nM) was incubated at 30°C in 40 mM Tris·HCl (pH 8.0), 50 mM KCl, and 100 mM MgCl₂ in the presence (lanes 1 and 2) or absence (lanes 3 and 4) of unlabeled RZ6 ribozyme (51) (37 μM) for 0 h (lanes 1 and 3) or 16 h (lanes 2 and 4) (12). Full-length SL1′ and the 3′ product (3′-P) were separated by denaturing PAGE and visualized with a PhosphorImager.

Fig. 2.

NMR structure of SL1′.(a) Stereoview of the heavy-atom superposition of the 10 lowest energy structures. (b) Minimized average structure of the active internal loop viewed from the minor groove (Upper) and down the helix axis (Lower). The phosphorus at the cleavage site is shown as a yellow sphere. (c) Base pair geometry for internal loop residues in the minimized average structure. The 2′-OH and amino protons are shown here in addition to the heavy atoms. The dashed lines represent potential hydrogen bonds based on short distances (<3.4 Å) observed in at least one structure. Internal loop nucleotides are colored according to the scheme presented in b.

Fig. 3.

Structural comparison of the inactive and active conformations of the VS ribozyme stem-loop I internal loop. (a) Summary of the base-pairing interactions in the inactive (Left) (13, 14) and active (Right) conformations. (b) Stereoview for comparing the inactive and active conformations. The inactive stem-loop I conformation (pastel colors) is the best representative conformer from the ensemble of NMR structures of Michiels et al. (ref. ; PDB ID code 1E4P), and the active conformation (darker colors) is the minimized average structure described here. The superposition was obtained by minimizing the rmsd for heavy atoms of residues C619–G623 and C637–G640. Internal loop nucleotides are colored according to the scheme presented in a.

Fig. 4.

Mg²⁺ binding sites of SL1′.(a) Schematic comparing the 5′-A G-3′/5′-C G-3′ metal-ion binding motif (box) of the hammerhead ribozyme (Left) (36) and the two 5′-A G-3′/5′-C G-3′ motifs (boxes) in the internal loop of SL1′ (Right). For the hammerhead ribozyme, the bound Mg²⁺ is represented by a red circle, and the cleavage site is indicated by the arrowhead. (b) Chemical-shift changes (Δ in ppm ± 0.03 ppm; see Methods) after addition of 10 mM MgCl₂ for the 1′, 2′, 3′, 6/8, and 2/5 proton and carbon atoms of all SL1′ residues. (c) Summary of Mg²⁺ binding data on the stereoview of the minimized average structure of SL1′ (residues C4-A9 and U16-G20). Significant chemical-shift changes after addition of 10 mM MgCl₂ (Δ >0.2 ppm) were mapped as green spheres on the respective C1′, C2′, C3′, C6/C8, and C2/C5 atoms. Putative locations for the Mg²⁺ (red spheres) were obtained by heavy-atom superposition of the 5′-A G-3′/5′-C G-3′ motif from the x-ray structure of the Mg²⁺-bound hammerhead ribozyme (only Mg²⁺ are shown) (36) with the 5′-A639 G640-3′/5′-C619 G 620-3′ (rmsd of 1.01 Å) and the 5′-A622 G623-3′/5′-C637 G 638-3′ (rmsd of 1.56 Å) motifs from the minimized average structure of SL1′.

Fig. 5.

The internal loop structure of the active stem-loop I is a tertiary interaction motif (a) Sequence and secondary structure of the pattern searched by mc-search and of helix 44 of 16S rRNA of T. thermophilus (40) (b) A superposition between helix 44 (PDB ID code 1FJG; pastel colors) and the minimized average structure of SL1′ (darker colors) was obtained by minimizing the rmsd for heavy atoms of the five residues in the internal loop (1.40 Å). Also shown are residues in helix 13 of 16S rRNA, which form a tertiary interaction with helix 44 (c) Summary of the base-pairing and stacking interactions in helices 13 and 44 and of tertiary contacts between them. Solid and dashed black lines indicate base pairs with two hydrogen bonds (either Watson–Crick or sheared G-A) and one hydrogen bond, respectively. Black rectangles indicate base stacking. Red spheres indicate riboses involved in ribose–ribose contacts (red dashed lines). The two adenines A1433 and A1434 participate in A-minor motifs (green dashed lines).