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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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Stabilization of the A730 loop by Mg2+ ions. Imino regions of 1D flip-back watergate (44,45) 1H spectra of SLVI collected at 15°C in NMR buffer containing different concentrations of free MgCl2. Imino proton assignments were derived from 2D NOESY spectra collected in NMR buffer at 0, 5 and 10 mM MgCl2.
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Figure 2: Stabilization of the A730 loop by Mg2+ ions. Imino regions of 1D flip-back watergate (44,45) 1H spectra of SLVI collected at 15°C in NMR buffer containing different concentrations of free MgCl2. Imino proton assignments were derived from 2D NOESY spectra collected in NMR buffer at 0, 5 and 10 mM MgCl2.

Mentions: Given that the VS ribozyme is active in the presence of Mg2+ ions (9) and that Mg2+-ion binding sites have been identified in the A730 loop domain (63), we first examined the effect of MgCl2 concentration on the folding of the SLVI RNA by 1D imino 1H NMR spectra. The 1D imino 1H NMR spectrum of the SLVI RNA recorded in the absence of Mg2+ ions shows signals of variable intensities characteristic of folded RNAs containing dynamic regions (Figure 2, bottom spectrum). These imino proton signals were assigned from a 2D NOESY spectrum collected under the same conditions. It was found that all imino protons of the SLVI RNA give an intense signals in this 1D spectrum, except for the G6 and G9 imino protons, which are not observed, and the U18 and G19 imino protons, which yield signals of weak intensities (Figure 2, bottom spectrum). These absent and weak imino proton signals indicate that the A730 loop domain is unstable in the absence of Mg2+ ions. Upon addition of Mg2+ ions, the imino signals of U18 and G19 become significantly more intense (Figure 2, top spectra) and a weak imino proton signal from G6 appears in the spectrum. Analysis of 2D NOESY and 2D HNN-COSY spectra collected at 5 mM MgCl2 confirms formation of all base pairs from the predicted secondary structure of the SLVI RNA (Figure 1C). Although the imino NMR data do not provide evidence for stabilization of non-canonical base pairs within the A730 loop, they indicate that the A730 loop domain is stabilized by Mg2+ ions, in agreement with previous biochemical studies. A MgCl2 concentration of 5 mM was found sufficient to produce the characteristic spectral changes (Figure 2) and, therefore, was selected for future NMR studies.Figure 2.


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)

Stabilization of the A730 loop by Mg2+ ions. Imino regions of 1D flip-back watergate (44,45) 1H spectra of SLVI collected at 15°C in NMR buffer containing different concentrations of free MgCl2. Imino proton assignments were derived from 2D NOESY spectra collected in NMR buffer at 0, 5 and 10 mM MgCl2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3105416&req=5

Figure 2: Stabilization of the A730 loop by Mg2+ ions. Imino regions of 1D flip-back watergate (44,45) 1H spectra of SLVI collected at 15°C in NMR buffer containing different concentrations of free MgCl2. Imino proton assignments were derived from 2D NOESY spectra collected in NMR buffer at 0, 5 and 10 mM MgCl2.
Mentions: Given that the VS ribozyme is active in the presence of Mg2+ ions (9) and that Mg2+-ion binding sites have been identified in the A730 loop domain (63), we first examined the effect of MgCl2 concentration on the folding of the SLVI RNA by 1D imino 1H NMR spectra. The 1D imino 1H NMR spectrum of the SLVI RNA recorded in the absence of Mg2+ ions shows signals of variable intensities characteristic of folded RNAs containing dynamic regions (Figure 2, bottom spectrum). These imino proton signals were assigned from a 2D NOESY spectrum collected under the same conditions. It was found that all imino protons of the SLVI RNA give an intense signals in this 1D spectrum, except for the G6 and G9 imino protons, which are not observed, and the U18 and G19 imino protons, which yield signals of weak intensities (Figure 2, bottom spectrum). These absent and weak imino proton signals indicate that the A730 loop domain is unstable in the absence of Mg2+ ions. Upon addition of Mg2+ ions, the imino signals of U18 and G19 become significantly more intense (Figure 2, top spectra) and a weak imino proton signal from G6 appears in the spectrum. Analysis of 2D NOESY and 2D HNN-COSY spectra collected at 5 mM MgCl2 confirms formation of all base pairs from the predicted secondary structure of the SLVI RNA (Figure 1C). Although the imino NMR data do not provide evidence for stabilization of non-canonical base pairs within the A730 loop, they indicate that the A730 loop domain is stabilized by Mg2+ ions, in agreement with previous biochemical studies. A MgCl2 concentration of 5 mM was found sufficient to produce the characteristic spectral changes (Figure 2) and, therefore, was selected for future NMR studies.Figure 2.

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