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NMR structure of the A730 loop of the Neurospora VS ribozyme: insights into the formation of the active site.

Desjardins G, Bonneau E, Girard N, Boisbouvier J, Legault P - Nucleic Acids Res. (2011)

Bottom Line: The S-turn appears necessary to expose the Watson-Crick edge of a catalytically important residue (A756) so that it can fulfill its role in catalysis.The A730 loop and the cleavage site loop of the VS ribozyme display structural similarities to internal loops found in the active site of the hairpin ribozyme.These similarities provided a rationale to build a model of the VS ribozyme active site based on the crystal structure of the hairpin ribozyme.

View Article: PubMed Central - PubMed

Affiliation: Département de Biochimie, Université de Montréal, CP 6128, Succursale Centre-Ville, Montréal, Québec H3C 3J7, Canada.

ABSTRACT
The Neurospora VS ribozyme is a small nucleolytic ribozyme with unique primary, secondary and global tertiary structures, which displays mechanistic similarities to the hairpin ribozyme. Here, we determined the high-resolution NMR structure of a stem-loop VI fragment containing the A730 internal loop, which forms part of the active site. In the presence of magnesium ions, the A730 loop adopts a structure that is consistent with existing biochemical data and most likely reflects its conformation in the VS ribozyme prior to docking with the cleavage site internal loop. Interestingly, the A730 loop adopts an S-turn motif that is also present in loop B within the hairpin ribozyme active site. The S-turn appears necessary to expose the Watson-Crick edge of a catalytically important residue (A756) so that it can fulfill its role in catalysis. The A730 loop and the cleavage site loop of the VS ribozyme display structural similarities to internal loops found in the active site of the hairpin ribozyme. These similarities provided a rationale to build a model of the VS ribozyme active site based on the crystal structure of the hairpin ribozyme.

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Homology modeling of the VS ribozyme active site. (A) Heavy atom superposition of the G638 and G620 nucleotides of the VS ribozyme with the G8 and A − 1 nucleotides of the hairpin ribozyme [pdb entry 1M5O; (60)]. (B) Modeling of the active site by association of the substrate internal loop and the A730 internal loop (see text). For simplicity only heavy atoms are shown and the ribbon replacing the phosphorus and non-bonded oxygen atoms is used to indicate the backbone topology. The yellow sphere represents the scissile phosphate.
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Figure 7: Homology modeling of the VS ribozyme active site. (A) Heavy atom superposition of the G638 and G620 nucleotides of the VS ribozyme with the G8 and A − 1 nucleotides of the hairpin ribozyme [pdb entry 1M5O; (60)]. (B) Modeling of the active site by association of the substrate internal loop and the A730 internal loop (see text). For simplicity only heavy atoms are shown and the ribbon replacing the phosphorus and non-bonded oxygen atoms is used to indicate the backbone topology. The yellow sphere represents the scissile phosphate.

Mentions: In the crystal structure of a transition state mimic of the hairpin ribozyme, the N1 of G8 is within hydrogen-bonding distance of the 2′-OH nucleophile, and its exocyclic amine forms a hydrogen bond with the scissile phosphate (60). A comparison between this X-ray structure of the hairpin ribozyme and the NMR structure of the cleavage site loop of the VS ribozyme (28) reveals that the G residue proposed to serve as the general base (G638 in the VS ribozyme and G8 in the hairpin ribozyme) and the N − 1 residue (G620 in the VS ribozyme and A − 1 in the hairpin ribozyme) form similar cross-strand purine stacks. The heavy atom superposition between these two sets of bases illustrates the similarity of these purine stacks (Figure 7A; rmsd of 0.8 Å). Thus, only small changes in the relative positioning of G638 with respect to G620 would be necessary to bring G638 in the proper orientation with respect to the cleavage site. For modeling purposes, we modified the positions of the G621 and G638 bases in the cleavage site internal loop structure to mimic the position of functionally equivalent residues in the hairpin ribozyme (Figure 7B).Figure 7.


NMR structure of the A730 loop of the Neurospora VS ribozyme: insights into the formation of the active site.

Desjardins G, Bonneau E, Girard N, Boisbouvier J, Legault P - Nucleic Acids Res. (2011)

Homology modeling of the VS ribozyme active site. (A) Heavy atom superposition of the G638 and G620 nucleotides of the VS ribozyme with the G8 and A − 1 nucleotides of the hairpin ribozyme [pdb entry 1M5O; (60)]. (B) Modeling of the active site by association of the substrate internal loop and the A730 internal loop (see text). For simplicity only heavy atoms are shown and the ribbon replacing the phosphorus and non-bonded oxygen atoms is used to indicate the backbone topology. The yellow sphere represents the scissile phosphate.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105416&req=5

Figure 7: Homology modeling of the VS ribozyme active site. (A) Heavy atom superposition of the G638 and G620 nucleotides of the VS ribozyme with the G8 and A − 1 nucleotides of the hairpin ribozyme [pdb entry 1M5O; (60)]. (B) Modeling of the active site by association of the substrate internal loop and the A730 internal loop (see text). For simplicity only heavy atoms are shown and the ribbon replacing the phosphorus and non-bonded oxygen atoms is used to indicate the backbone topology. The yellow sphere represents the scissile phosphate.
Mentions: In the crystal structure of a transition state mimic of the hairpin ribozyme, the N1 of G8 is within hydrogen-bonding distance of the 2′-OH nucleophile, and its exocyclic amine forms a hydrogen bond with the scissile phosphate (60). A comparison between this X-ray structure of the hairpin ribozyme and the NMR structure of the cleavage site loop of the VS ribozyme (28) reveals that the G residue proposed to serve as the general base (G638 in the VS ribozyme and G8 in the hairpin ribozyme) and the N − 1 residue (G620 in the VS ribozyme and A − 1 in the hairpin ribozyme) form similar cross-strand purine stacks. The heavy atom superposition between these two sets of bases illustrates the similarity of these purine stacks (Figure 7A; rmsd of 0.8 Å). Thus, only small changes in the relative positioning of G638 with respect to G620 would be necessary to bring G638 in the proper orientation with respect to the cleavage site. For modeling purposes, we modified the positions of the G621 and G638 bases in the cleavage site internal loop structure to mimic the position of functionally equivalent residues in the hairpin ribozyme (Figure 7B).Figure 7.

Bottom Line: The S-turn appears necessary to expose the Watson-Crick edge of a catalytically important residue (A756) so that it can fulfill its role in catalysis.The A730 loop and the cleavage site loop of the VS ribozyme display structural similarities to internal loops found in the active site of the hairpin ribozyme.These similarities provided a rationale to build a model of the VS ribozyme active site based on the crystal structure of the hairpin ribozyme.

View Article: PubMed Central - PubMed

Affiliation: Département de Biochimie, Université de Montréal, CP 6128, Succursale Centre-Ville, Montréal, Québec H3C 3J7, Canada.

ABSTRACT
The Neurospora VS ribozyme is a small nucleolytic ribozyme with unique primary, secondary and global tertiary structures, which displays mechanistic similarities to the hairpin ribozyme. Here, we determined the high-resolution NMR structure of a stem-loop VI fragment containing the A730 internal loop, which forms part of the active site. In the presence of magnesium ions, the A730 loop adopts a structure that is consistent with existing biochemical data and most likely reflects its conformation in the VS ribozyme prior to docking with the cleavage site internal loop. Interestingly, the A730 loop adopts an S-turn motif that is also present in loop B within the hairpin ribozyme active site. The S-turn appears necessary to expose the Watson-Crick edge of a catalytically important residue (A756) so that it can fulfill its role in catalysis. The A730 loop and the cleavage site loop of the VS ribozyme display structural similarities to internal loops found in the active site of the hairpin ribozyme. These similarities provided a rationale to build a model of the VS ribozyme active site based on the crystal structure of the hairpin ribozyme.

Show MeSH