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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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Determination of adenine pKa’s in SLVI. (A) Superposition of the aromatic C2–H2 regions of 2D 1H–13C HMQC spectra collected at 25°C and at pH 4.7 (beige), pH 5.1 (black), pH 5.5 (pale blue) and pH 8.6 (grayish blue). Arrows point to significant pH-dependent changes in 13C chemical shift for A8 and A20. (B) Summary of the adenine pKa values in the A730 internal loop.
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Figure 6: Determination of adenine pKa’s in SLVI. (A) Superposition of the aromatic C2–H2 regions of 2D 1H–13C HMQC spectra collected at 25°C and at pH 4.7 (beige), pH 5.1 (black), pH 5.5 (pale blue) and pH 8.6 (grayish blue). Arrows point to significant pH-dependent changes in 13C chemical shift for A8 and A20. (B) Summary of the adenine pKa values in the A730 internal loop.

Mentions: Given the predicted role of A756 as a general acid in the cleavage reaction, we were interested to determine if the structure of the A730 loop imparts a shifted pKa value for A756. Hence, we determined the pKa of adenines in the A730 loop of SLVI by 13C NMR methods. It has been previously shown that the C2 chemical shift of AMP undergoes an 8-ppm upfield displacement upon protonation at its N1 position (73), and pH-dependent change in C2 chemical shifts have been used to determine adenine pKa’s in folded RNAs (27,29,36–39,73). In SLVI, only two C2 resonances are significantly affected by pH; the A8 and A20 C2 resonances are shifted upfield by 2.4 and 3.3 ppm, respectively, when the pH is decreased from 8.6 to 4.7 (Figure 6A). For both A8 and A20, a single C2–H2 crosspeak is observed in this pH range, indicating fast exchange dynamics on the NMR chemical shift timescale. In such cases, the change in C2 chemical shift at a given pH value can be used to derive pKa values. We obtained a pKa of 4.44 ± 0.10 for A8 and a pKa of 4.74 ± 0.12 for A20 (Figure 6B and Supplementary Figure S1). These pKa values are slightly higher than the pKa value of adenine in single-stranded RNA [∼3.7; (74)] and are compatible with the accessibility of the N1 positions of A8 and A20 in the SLVI structure. Local electronic effects, such as sequence context, base stacking and Mg2+ binding could be responsible for these small pKa shifts. However, there is a discrepancy of about one pH unit between the pKa of A8 and the catalytic pKa [between 5.2 and 5.8; (23,25)]. Such discrepancy is not surprising, since it is expected that formation of the active site will change the chemical environment of the A756 base and thus, affect its pKa.Figure 6.


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)

Determination of adenine pKa’s in SLVI. (A) Superposition of the aromatic C2–H2 regions of 2D 1H–13C HMQC spectra collected at 25°C and at pH 4.7 (beige), pH 5.1 (black), pH 5.5 (pale blue) and pH 8.6 (grayish blue). Arrows point to significant pH-dependent changes in 13C chemical shift for A8 and A20. (B) Summary of the adenine pKa values in the A730 internal loop.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 6: Determination of adenine pKa’s in SLVI. (A) Superposition of the aromatic C2–H2 regions of 2D 1H–13C HMQC spectra collected at 25°C and at pH 4.7 (beige), pH 5.1 (black), pH 5.5 (pale blue) and pH 8.6 (grayish blue). Arrows point to significant pH-dependent changes in 13C chemical shift for A8 and A20. (B) Summary of the adenine pKa values in the A730 internal loop.
Mentions: Given the predicted role of A756 as a general acid in the cleavage reaction, we were interested to determine if the structure of the A730 loop imparts a shifted pKa value for A756. Hence, we determined the pKa of adenines in the A730 loop of SLVI by 13C NMR methods. It has been previously shown that the C2 chemical shift of AMP undergoes an 8-ppm upfield displacement upon protonation at its N1 position (73), and pH-dependent change in C2 chemical shifts have been used to determine adenine pKa’s in folded RNAs (27,29,36–39,73). In SLVI, only two C2 resonances are significantly affected by pH; the A8 and A20 C2 resonances are shifted upfield by 2.4 and 3.3 ppm, respectively, when the pH is decreased from 8.6 to 4.7 (Figure 6A). For both A8 and A20, a single C2–H2 crosspeak is observed in this pH range, indicating fast exchange dynamics on the NMR chemical shift timescale. In such cases, the change in C2 chemical shift at a given pH value can be used to derive pKa values. We obtained a pKa of 4.44 ± 0.10 for A8 and a pKa of 4.74 ± 0.12 for A20 (Figure 6B and Supplementary Figure S1). These pKa values are slightly higher than the pKa value of adenine in single-stranded RNA [∼3.7; (74)] and are compatible with the accessibility of the N1 positions of A8 and A20 in the SLVI structure. Local electronic effects, such as sequence context, base stacking and Mg2+ binding could be responsible for these small pKa shifts. However, there is a discrepancy of about one pH unit between the pKa of A8 and the catalytic pKa [between 5.2 and 5.8; (23,25)]. Such discrepancy is not surprising, since it is expected that formation of the active site will change the chemical environment of the A756 base and thus, affect its pKa.Figure 6.

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