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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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Formation of a cis WC/WC G9-A20 base pair in the A730 loop. (A) Selected regions from a 2D 1H–15N CPMG-NOESY spectrum showing NOEs that define the geometry of the G9–A20 base pair. The spectrum was collected at 15°C with a mixing time of 160 ms. (B) The G9–A20 base pair in the 20 lowest-energy structures. The superposition is from Figure 3a. (C) Stacking of the G9–A20 base pair onto the C10–G19 base pair in the lowest-energy structure of SLVI. Dashed lines connect protons for which a NOE is observed in (A).
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Figure 4: Formation of a cis WC/WC G9-A20 base pair in the A730 loop. (A) Selected regions from a 2D 1H–15N CPMG-NOESY spectrum showing NOEs that define the geometry of the G9–A20 base pair. The spectrum was collected at 15°C with a mixing time of 160 ms. (B) The G9–A20 base pair in the 20 lowest-energy structures. The superposition is from Figure 3a. (C) Stacking of the G9–A20 base pair onto the C10–G19 base pair in the lowest-energy structure of SLVI. Dashed lines connect protons for which a NOE is observed in (A).

Mentions: The evidence for a cis-WC/WC G–A base pair in the A730 loop was obtained from 2D 1H–15N CMPG-NOESY and 3D 13C-edited HMQC-NOESY spectra. A strong NOE signal was observed between the A20 H2 and the G9 NH2 protons, which is typical of the cis-WC/WC G–A base pair geometry (Figure 4A). Several NOE signals were also observed that indicate stacking of the G9–A20 base pair on the C10–G19 base pair, including NOEs between A20 H2 and G19 NH and between A20 H1′ and G19 NH (Figure 4A). Initial structural calculations performed without specific hydrogen-bond restraints for the G9–A20 base pair revealed a cis-WC/WC G–A base pair geometry, thus, hydrogen-bond restraints defining this geometry were included in subsequent rounds of structural calculations. The superposition of the 20 lowest-energy structures show that this G–A base pair is well defined by the NMR data, although two conformations with different propeller twists and buckles are observed in the ensemble of structure (Figure 4B). These two alternative conformations observed in the NMR structures may reflect insufficient NMR restraints and/or conformational dynamics for this base pair. The absence of a detectable imino proton signal for G9 (Figure 2) supports conformational dynamics for the G9–A20 base pair.Figure 4.


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)

Formation of a cis WC/WC G9-A20 base pair in the A730 loop. (A) Selected regions from a 2D 1H–15N CPMG-NOESY spectrum showing NOEs that define the geometry of the G9–A20 base pair. The spectrum was collected at 15°C with a mixing time of 160 ms. (B) The G9–A20 base pair in the 20 lowest-energy structures. The superposition is from Figure 3a. (C) Stacking of the G9–A20 base pair onto the C10–G19 base pair in the lowest-energy structure of SLVI. Dashed lines connect protons for which a NOE is observed in (A).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 4: Formation of a cis WC/WC G9-A20 base pair in the A730 loop. (A) Selected regions from a 2D 1H–15N CPMG-NOESY spectrum showing NOEs that define the geometry of the G9–A20 base pair. The spectrum was collected at 15°C with a mixing time of 160 ms. (B) The G9–A20 base pair in the 20 lowest-energy structures. The superposition is from Figure 3a. (C) Stacking of the G9–A20 base pair onto the C10–G19 base pair in the lowest-energy structure of SLVI. Dashed lines connect protons for which a NOE is observed in (A).
Mentions: The evidence for a cis-WC/WC G–A base pair in the A730 loop was obtained from 2D 1H–15N CMPG-NOESY and 3D 13C-edited HMQC-NOESY spectra. A strong NOE signal was observed between the A20 H2 and the G9 NH2 protons, which is typical of the cis-WC/WC G–A base pair geometry (Figure 4A). Several NOE signals were also observed that indicate stacking of the G9–A20 base pair on the C10–G19 base pair, including NOEs between A20 H2 and G19 NH and between A20 H1′ and G19 NH (Figure 4A). Initial structural calculations performed without specific hydrogen-bond restraints for the G9–A20 base pair revealed a cis-WC/WC G–A base pair geometry, thus, hydrogen-bond restraints defining this geometry were included in subsequent rounds of structural calculations. The superposition of the 20 lowest-energy structures show that this G–A base pair is well defined by the NMR data, although two conformations with different propeller twists and buckles are observed in the ensemble of structure (Figure 4B). These two alternative conformations observed in the NMR structures may reflect insufficient NMR restraints and/or conformational dynamics for this base pair. The absence of a detectable imino proton signal for G9 (Figure 2) supports conformational dynamics for the G9–A20 base pair.Figure 4.

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