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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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NMR solution structure of the SLVI RNA fragment. (A) Stereoview of the 20 lowest-energy structures. The superposition was made on the minimized average structure (not shown) over heavy atoms of residues 2–25. The view is into the minor groove of the A730 active site internal loop. (B and C) Stick representations of the lowest-energy structure of SLVI. 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. SLVI nucleotides are color-coded: the loop closing base pairs (G6–C21 and C10–G19) are dark gray, C7 (C755) is magenta, A8 (A756) is green, G9 (G757) is gold and A20 (A730) is blue.
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Figure 3: NMR solution structure of the SLVI RNA fragment. (A) Stereoview of the 20 lowest-energy structures. The superposition was made on the minimized average structure (not shown) over heavy atoms of residues 2–25. The view is into the minor groove of the A730 active site internal loop. (B and C) Stick representations of the lowest-energy structure of SLVI. 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. SLVI nucleotides are color-coded: the loop closing base pairs (G6–C21 and C10–G19) are dark gray, C7 (C755) is magenta, A8 (A756) is green, G9 (G757) is gold and A20 (A730) is blue.

Mentions: The structure of the SLVI RNA was obtained by multidimensional heteronuclear NMR methods. Complete resonance assignments were obtained for all observable 1H, 13C and 15N atoms of SLVI (Supplementary Table S1). The structure determination included NOE-derived distance restraints, dihedral angle restraints and RDC restraints (Table 1). The SLVI structure is well defined by the NMR data with a heavy atom rmsd of 0.67 ± 0.17 Å for the 20 lowest-energy structures (residues 2–25; Table 1 and Figure 3A). The SLVI RNA forms a hairpin structure containing a 5′–3′ stem (residues 2–5 and 22–25; rmsd of 0.53 ± 0.07 Å), a hairpin stem (residues 11–18; rmsd of 0.31 ± 0.01 Å), and the A730 loop (residues 6–10 and 19–21; rmsd of 0.65 ± 0.24 Å). Stem regions form regular A-form helices and the GAAA tetraloop adopts the typical GNRA fold with its characteristic sheared G–A base pair and 3′-purine stack (64,65). The A730 loop domain imparts a ∼150° interhelical angle between the two stems of SLVI and contains two structural characteristics that were not previously identified: a cis-WC/WC G9–A20 base pair and an S-turn motif that protrudes C7 and A8 in the minor groove.Figure 3.


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)

NMR solution structure of the SLVI RNA fragment. (A) Stereoview of the 20 lowest-energy structures. The superposition was made on the minimized average structure (not shown) over heavy atoms of residues 2–25. The view is into the minor groove of the A730 active site internal loop. (B and C) Stick representations of the lowest-energy structure of SLVI. 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. SLVI nucleotides are color-coded: the loop closing base pairs (G6–C21 and C10–G19) are dark gray, C7 (C755) is magenta, A8 (A756) is green, G9 (G757) is gold and A20 (A730) is blue.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: NMR solution structure of the SLVI RNA fragment. (A) Stereoview of the 20 lowest-energy structures. The superposition was made on the minimized average structure (not shown) over heavy atoms of residues 2–25. The view is into the minor groove of the A730 active site internal loop. (B and C) Stick representations of the lowest-energy structure of SLVI. 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. SLVI nucleotides are color-coded: the loop closing base pairs (G6–C21 and C10–G19) are dark gray, C7 (C755) is magenta, A8 (A756) is green, G9 (G757) is gold and A20 (A730) is blue.
Mentions: The structure of the SLVI RNA was obtained by multidimensional heteronuclear NMR methods. Complete resonance assignments were obtained for all observable 1H, 13C and 15N atoms of SLVI (Supplementary Table S1). The structure determination included NOE-derived distance restraints, dihedral angle restraints and RDC restraints (Table 1). The SLVI structure is well defined by the NMR data with a heavy atom rmsd of 0.67 ± 0.17 Å for the 20 lowest-energy structures (residues 2–25; Table 1 and Figure 3A). The SLVI RNA forms a hairpin structure containing a 5′–3′ stem (residues 2–5 and 22–25; rmsd of 0.53 ± 0.07 Å), a hairpin stem (residues 11–18; rmsd of 0.31 ± 0.01 Å), and the A730 loop (residues 6–10 and 19–21; rmsd of 0.65 ± 0.24 Å). Stem regions form regular A-form helices and the GAAA tetraloop adopts the typical GNRA fold with its characteristic sheared G–A base pair and 3′-purine stack (64,65). The A730 loop domain imparts a ∼150° interhelical angle between the two stems of SLVI and contains two structural characteristics that were not previously identified: a cis-WC/WC G9–A20 base pair and an S-turn motif that protrudes C7 and A8 in the minor groove.Figure 3.

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
Related in: MedlinePlus