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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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S-turn motif in the A730 loop of the SLVI RNA. (A) Schematic summarizing the inter-residue NOEs for the A730 internal loop of the SLVI RNA. Black lines indicate NOEs between nucleotides that are adjacent in the sequence, pink lines indicate NOEs between base-pairing residues, blue lines indicate NOEs between G6 and G9, and orange lines refer to NOEs between C7 and G9. For simplicity, all ribose protons (H1′, H2′, H3′, H4′, H5′ and H5″) were grouped under the ribose denomination. (B and C) Close up views of the S-turn motif in the lowest-energy structure showing (B) the ribose reversal at A8 and nearby phosphates and (C) stacking of C7 and A8 in the minor groove and stabilizing hydrogen bonds. In (B) the pro–Rp oxygens are shown in blue and the 2′-oxygens in red. In (C) three hydrogen bonds are shown (A8 N3: G9 2′–OH, C7 NH2: G9 N3 and A8 2′–OH: G9 O4′) that likely stabilize the S-turn motif. 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.
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Figure 5: S-turn motif in the A730 loop of the SLVI RNA. (A) Schematic summarizing the inter-residue NOEs for the A730 internal loop of the SLVI RNA. Black lines indicate NOEs between nucleotides that are adjacent in the sequence, pink lines indicate NOEs between base-pairing residues, blue lines indicate NOEs between G6 and G9, and orange lines refer to NOEs between C7 and G9. For simplicity, all ribose protons (H1′, H2′, H3′, H4′, H5′ and H5″) were grouped under the ribose denomination. (B and C) Close up views of the S-turn motif in the lowest-energy structure showing (B) the ribose reversal at A8 and nearby phosphates and (C) stacking of C7 and A8 in the minor groove and stabilizing hydrogen bonds. In (B) the pro–Rp oxygens are shown in blue and the 2′-oxygens in red. In (C) three hydrogen bonds are shown (A8 N3: G9 2′–OH, C7 NH2: G9 N3 and A8 2′–OH: G9 O4′) that likely stabilize the S-turn motif. 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.

Mentions: The structure of the A730 loop domain is defined by a large number of NOEs, including several unusual sequential NOEs in the G6–G9 stretch and non-sequential NOEs between nucleotides G6 and G9 (Figure 5A). These NOEs are consistent with the unusual ribose-phosphate backbone of the A730 loop domain, which adopts an S-turn between nucleotides G6 and C10. The S-turn is a common RNA motif, first structurally identified in the loop E of eukaryotic 5S rRNA (66) and the sarcin–ricin loop of 28S rRNA (67), but since found in other structural contexts (68–72). In the A730 loop, the S-turn is created by ribose reversal at A8, with its 2′-OH group pointing in a direction opposite to the 2′-OH groups of adjacent nucleotides (Figure 5B). In the majority of the lowest-energy structures (17/20), the ribose of A8 adopts a 2′-endo conformation, a characteristic of an S-turn, which is in agreement with the intense H1′–H2′ signal in the DQF-COSY spectrum (not shown). The S-turn of the A730 loop leads to bulging out of both the C7 and A8 residues with their Watson–Crick edges exposed in the minor groove. The adjacent G9–A20 base pair possesses a larger C1′–C1′ distance than standard Watson–Crick base pairs that likely helps stabilize the S-turn. Three hydrogen bonds involving G9 and the protruded C7 and A8 bases are found in the ensemble of 20 lowest-energy structures and connect: (i) G9 O4′ and A8 O2′ (2.6 ± 0.4 Å); (ii) G9 O2′ and A8 N3 (3.3 ± 1.0 Å) and (iii) G9 N3 and C7 N4 (3.4 ± 0.8 Å; Figure 5C). Interestingly, an S-turn motif has also been previously found in loop B of the hairpin ribozyme (70), suggesting that it may be important for catalysis by the VS ribozyme (see discussion).Figure 5.


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)

S-turn motif in the A730 loop of the SLVI RNA. (A) Schematic summarizing the inter-residue NOEs for the A730 internal loop of the SLVI RNA. Black lines indicate NOEs between nucleotides that are adjacent in the sequence, pink lines indicate NOEs between base-pairing residues, blue lines indicate NOEs between G6 and G9, and orange lines refer to NOEs between C7 and G9. For simplicity, all ribose protons (H1′, H2′, H3′, H4′, H5′ and H5″) were grouped under the ribose denomination. (B and C) Close up views of the S-turn motif in the lowest-energy structure showing (B) the ribose reversal at A8 and nearby phosphates and (C) stacking of C7 and A8 in the minor groove and stabilizing hydrogen bonds. In (B) the pro–Rp oxygens are shown in blue and the 2′-oxygens in red. In (C) three hydrogen bonds are shown (A8 N3: G9 2′–OH, C7 NH2: G9 N3 and A8 2′–OH: G9 O4′) that likely stabilize the S-turn motif. 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.
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Related In: Results  -  Collection

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Figure 5: S-turn motif in the A730 loop of the SLVI RNA. (A) Schematic summarizing the inter-residue NOEs for the A730 internal loop of the SLVI RNA. Black lines indicate NOEs between nucleotides that are adjacent in the sequence, pink lines indicate NOEs between base-pairing residues, blue lines indicate NOEs between G6 and G9, and orange lines refer to NOEs between C7 and G9. For simplicity, all ribose protons (H1′, H2′, H3′, H4′, H5′ and H5″) were grouped under the ribose denomination. (B and C) Close up views of the S-turn motif in the lowest-energy structure showing (B) the ribose reversal at A8 and nearby phosphates and (C) stacking of C7 and A8 in the minor groove and stabilizing hydrogen bonds. In (B) the pro–Rp oxygens are shown in blue and the 2′-oxygens in red. In (C) three hydrogen bonds are shown (A8 N3: G9 2′–OH, C7 NH2: G9 N3 and A8 2′–OH: G9 O4′) that likely stabilize the S-turn motif. 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.
Mentions: The structure of the A730 loop domain is defined by a large number of NOEs, including several unusual sequential NOEs in the G6–G9 stretch and non-sequential NOEs between nucleotides G6 and G9 (Figure 5A). These NOEs are consistent with the unusual ribose-phosphate backbone of the A730 loop domain, which adopts an S-turn between nucleotides G6 and C10. The S-turn is a common RNA motif, first structurally identified in the loop E of eukaryotic 5S rRNA (66) and the sarcin–ricin loop of 28S rRNA (67), but since found in other structural contexts (68–72). In the A730 loop, the S-turn is created by ribose reversal at A8, with its 2′-OH group pointing in a direction opposite to the 2′-OH groups of adjacent nucleotides (Figure 5B). In the majority of the lowest-energy structures (17/20), the ribose of A8 adopts a 2′-endo conformation, a characteristic of an S-turn, which is in agreement with the intense H1′–H2′ signal in the DQF-COSY spectrum (not shown). The S-turn of the A730 loop leads to bulging out of both the C7 and A8 residues with their Watson–Crick edges exposed in the minor groove. The adjacent G9–A20 base pair possesses a larger C1′–C1′ distance than standard Watson–Crick base pairs that likely helps stabilize the S-turn. Three hydrogen bonds involving G9 and the protruded C7 and A8 bases are found in the ensemble of 20 lowest-energy structures and connect: (i) G9 O4′ and A8 O2′ (2.6 ± 0.4 Å); (ii) G9 O2′ and A8 N3 (3.3 ± 1.0 Å) and (iii) G9 N3 and C7 N4 (3.4 ± 0.8 Å; Figure 5C). Interestingly, an S-turn motif has also been previously found in loop B of the hairpin ribozyme (70), suggesting that it may be important for catalysis by the VS ribozyme (see discussion).Figure 5.

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