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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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The Neurospora VS ribozyme and its A730 internal loop. (A) Primary and secondary structures of the VS ribozyme (wild-type sequence nucleotides 617–783). The site of self-cleavage is indicated by an arrow, and circled nucleotides in loops I and V form the I/V kissing-loop interaction. (B) Schematic of the VS ribozyme (left) and hairpin ribozyme (right) illustrating similarities at the active site (see text). The residues highlighted with white circles are key players of proposed general acid-based mechanisms (4). (C) Primary and predicted secondary structures of the 26-nt SLVI RNA fragment, which includes the A730 loop domain (gray box). Phosphate groups that display inhibitory effect when substituted by Rp phosphorothioate are indicated by an arrow (20,63), and the arrow is filled in those cases where the inhibition could be suppressed by addition of manganese ions (63).
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Figure 1: The Neurospora VS ribozyme and its A730 internal loop. (A) Primary and secondary structures of the VS ribozyme (wild-type sequence nucleotides 617–783). The site of self-cleavage is indicated by an arrow, and circled nucleotides in loops I and V form the I/V kissing-loop interaction. (B) Schematic of the VS ribozyme (left) and hairpin ribozyme (right) illustrating similarities at the active site (see text). The residues highlighted with white circles are key players of proposed general acid-based mechanisms (4). (C) Primary and predicted secondary structures of the 26-nt SLVI RNA fragment, which includes the A730 loop domain (gray box). Phosphate groups that display inhibitory effect when substituted by Rp phosphorothioate are indicated by an arrow (20,63), and the arrow is filled in those cases where the inhibition could be suppressed by addition of manganese ions (63).

Mentions: The Neurospora VS ribozyme was first identified as a satellite RNA in the Varkud-1c strain and a few other isolates of Neurospora (6). A contiguous sequence of 154 nt was shown to be minimally required for self-cleavage in vitro (7). Its secondary structure contains six helical domains: stem–loop I (SLI) forms the substrate and stem–loops II–VI (SLII–SLVI) define the catalytic domain [Figure 1A; (8)]. The VS ribozyme is most active in the presence of divalent ions (9,10). Mg2+ ions allow tertiary-structure formation (8,11,12) and partly contribute to the chemistry of the cleavage reaction (10). Monovalent cations also support cleavage, albeit at a lower rate (10). The self-cleavage reaction takes place when the SLI substrate is located either at the 5′- or 3′-end of the catalytic domain (7,13), and, alternatively, trans-cleavage occurs when the substrate is synthesized separately from the catalytic domain (14). Substrate recognition involves formation of a kissing-loop interaction between loop I of the substrate and loop V of the catalytic domain (11). Formation of this I/V kissing-loop interaction is accompanied by a conformational change in SLI from an unshifted inactive conformation to a shifted active conformation (15). Our present understanding is that, in order for cleavage to occur, the cleavage site internal loop of SLI must dock in a cleft formed by SLII and SLVI to allow its interaction with the A730 loop of SLVI (4,12,16–25).Figure 1.


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)

The Neurospora VS ribozyme and its A730 internal loop. (A) Primary and secondary structures of the VS ribozyme (wild-type sequence nucleotides 617–783). The site of self-cleavage is indicated by an arrow, and circled nucleotides in loops I and V form the I/V kissing-loop interaction. (B) Schematic of the VS ribozyme (left) and hairpin ribozyme (right) illustrating similarities at the active site (see text). The residues highlighted with white circles are key players of proposed general acid-based mechanisms (4). (C) Primary and predicted secondary structures of the 26-nt SLVI RNA fragment, which includes the A730 loop domain (gray box). Phosphate groups that display inhibitory effect when substituted by Rp phosphorothioate are indicated by an arrow (20,63), and the arrow is filled in those cases where the inhibition could be suppressed by addition of manganese ions (63).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: The Neurospora VS ribozyme and its A730 internal loop. (A) Primary and secondary structures of the VS ribozyme (wild-type sequence nucleotides 617–783). The site of self-cleavage is indicated by an arrow, and circled nucleotides in loops I and V form the I/V kissing-loop interaction. (B) Schematic of the VS ribozyme (left) and hairpin ribozyme (right) illustrating similarities at the active site (see text). The residues highlighted with white circles are key players of proposed general acid-based mechanisms (4). (C) Primary and predicted secondary structures of the 26-nt SLVI RNA fragment, which includes the A730 loop domain (gray box). Phosphate groups that display inhibitory effect when substituted by Rp phosphorothioate are indicated by an arrow (20,63), and the arrow is filled in those cases where the inhibition could be suppressed by addition of manganese ions (63).
Mentions: The Neurospora VS ribozyme was first identified as a satellite RNA in the Varkud-1c strain and a few other isolates of Neurospora (6). A contiguous sequence of 154 nt was shown to be minimally required for self-cleavage in vitro (7). Its secondary structure contains six helical domains: stem–loop I (SLI) forms the substrate and stem–loops II–VI (SLII–SLVI) define the catalytic domain [Figure 1A; (8)]. The VS ribozyme is most active in the presence of divalent ions (9,10). Mg2+ ions allow tertiary-structure formation (8,11,12) and partly contribute to the chemistry of the cleavage reaction (10). Monovalent cations also support cleavage, albeit at a lower rate (10). The self-cleavage reaction takes place when the SLI substrate is located either at the 5′- or 3′-end of the catalytic domain (7,13), and, alternatively, trans-cleavage occurs when the substrate is synthesized separately from the catalytic domain (14). Substrate recognition involves formation of a kissing-loop interaction between loop I of the substrate and loop V of the catalytic domain (11). Formation of this I/V kissing-loop interaction is accompanied by a conformational change in SLI from an unshifted inactive conformation to a shifted active conformation (15). Our present understanding is that, in order for cleavage to occur, the cleavage site internal loop of SLI must dock in a cleft formed by SLII and SLVI to allow its interaction with the A730 loop of SLVI (4,12,16–25).Figure 1.

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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