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Helix-length compensation studies reveal the adaptability of the VS ribozyme architecture.

Lacroix-Labonté J, Girard N, Lemieux S, Legault P - Nucleic Acids Res. (2011)

Bottom Line: Several active substrate/ribozyme pairs were identified, indicating the presence of limited substrate promiscuity for stem Ib variants and helix-length compensation between stems Ib and V. 3D models of the I/V interaction were generated that are compatible with the kinetic data.These models further illustrate the adaptability of the VS ribozyme architecture for substrate cleavage and provide global structural information on the I/V kissing-loop interaction.By exploring higher-order compensatory mutations in RNA our approach brings a deeper understanding of the adaptability of RNA structure, while opening new avenues for RNA research.

View Article: PubMed Central - PubMed

Affiliation: Département de Biochimie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC, Canada.

ABSTRACT
Compensatory mutations in RNA are generally regarded as those that maintain base pairing, and their identification forms the basis of phylogenetic predictions of RNA secondary structure. However, other types of compensatory mutations can provide higher-order structural and evolutionary information. Here, we present a helix-length compensation study for investigating structure-function relationships in RNA. The approach is demonstrated for stem-loop I and stem-loop V of the Neurospora VS ribozyme, which form a kissing-loop interaction important for substrate recognition. To rapidly characterize the substrate specificity (k(cat)/K(M)) of several substrate/ribozyme pairs, a procedure was established for simultaneous kinetic characterization of multiple substrates. Several active substrate/ribozyme pairs were identified, indicating the presence of limited substrate promiscuity for stem Ib variants and helix-length compensation between stems Ib and V. 3D models of the I/V interaction were generated that are compatible with the kinetic data. These models further illustrate the adaptability of the VS ribozyme architecture for substrate cleavage and provide global structural information on the I/V kissing-loop interaction. By exploring higher-order compensatory mutations in RNA our approach brings a deeper understanding of the adaptability of RNA structure, while opening new avenues for RNA research.

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Model of an SLI/SLV complex compatible the kinetic data (common-core group 13). (A) Stick representation of the S0/R0 sub-model. (B) Cartoon representation of a superposition of the five variant S/R0 sub-models on the S0/R0 sub-model. The black arrow points at the scissile phosphate of S0/R0. The scissile phosphates of the following substrates are shown from bottom to top: S−1, S0, S+1, S+2, S+3, S+4. (C) Top-down and (D) front views of the superposition of the most catalytically efficient sub-models of each ribozyme. The following S/R pairs are shown from left to right: S−1/R+1, S0/R0, S+1/R−1, S+1/R−2, S+2/R−1, S+3/R−4. In (B), (C) and (D), the sub-models were superposed to the S0/R0 sub-model based on heavy atom alignment of the first base pair of SLV (residues 689 and 707 in S0/R0). SLI and SLV are represented in deep blue and orange, respectively. Helical axes are represented as red rods and the phosphorus atom at the cleavage site is shown as a sphere. The phosphorus atoms are color-coded according to the catalytic efficiency of the sub-models {[(kcat/KM)/(kcat/KM) ≥ 0.25]: deep orange; [0.25 > (kcat/KM)/(kcat/KM) > 0.01]: light orange and [(kcat/KM)/(kcat/KM) ≤ 0.01]: white}.
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gkr1018-F5: Model of an SLI/SLV complex compatible the kinetic data (common-core group 13). (A) Stick representation of the S0/R0 sub-model. (B) Cartoon representation of a superposition of the five variant S/R0 sub-models on the S0/R0 sub-model. The black arrow points at the scissile phosphate of S0/R0. The scissile phosphates of the following substrates are shown from bottom to top: S−1, S0, S+1, S+2, S+3, S+4. (C) Top-down and (D) front views of the superposition of the most catalytically efficient sub-models of each ribozyme. The following S/R pairs are shown from left to right: S−1/R+1, S0/R0, S+1/R−1, S+1/R−2, S+2/R−1, S+3/R−4. In (B), (C) and (D), the sub-models were superposed to the S0/R0 sub-model based on heavy atom alignment of the first base pair of SLV (residues 689 and 707 in S0/R0). SLI and SLV are represented in deep blue and orange, respectively. Helical axes are represented as red rods and the phosphorus atom at the cleavage site is shown as a sphere. The phosphorus atoms are color-coded according to the catalytic efficiency of the sub-models {[(kcat/KM)/(kcat/KM) ≥ 0.25]: deep orange; [0.25 > (kcat/KM)/(kcat/KM) > 0.01]: light orange and [(kcat/KM)/(kcat/KM) ≤ 0.01]: white}.

Mentions: The 3D structure of the S0/R0 sub-model from the selected group reveals that, although the helical axes of stems Ib and V are more or less colinear, the two stems are not coaxial (Figure 5A). Rather, there is a significant perpendicular displacement (dI−V ~ 20 Å) between these axes that results from the structure of the kissing-loop junction. The spatial relation between these two axes is reflected in the structure of variant S/R pairs, as illustrated in Figure 5B–D. The superposition of all S/R pairs containing R0 shows that the scissile phosphates of the high-activity substrates (deep orange spheres in Figure 5B) cover a ~7.5 Å distance on one side of stem Ib, whereas those of low-activity substrates (white spheres in Figure 5B) rotate around the helical axis towards a different side of the helix. Thus, this superposition indicates that limited helical displacements of the scissile phosphate are compatible with productive docking of the substrate internal loop with the catalytic domain.Figure 5.


Helix-length compensation studies reveal the adaptability of the VS ribozyme architecture.

Lacroix-Labonté J, Girard N, Lemieux S, Legault P - Nucleic Acids Res. (2011)

Model of an SLI/SLV complex compatible the kinetic data (common-core group 13). (A) Stick representation of the S0/R0 sub-model. (B) Cartoon representation of a superposition of the five variant S/R0 sub-models on the S0/R0 sub-model. The black arrow points at the scissile phosphate of S0/R0. The scissile phosphates of the following substrates are shown from bottom to top: S−1, S0, S+1, S+2, S+3, S+4. (C) Top-down and (D) front views of the superposition of the most catalytically efficient sub-models of each ribozyme. The following S/R pairs are shown from left to right: S−1/R+1, S0/R0, S+1/R−1, S+1/R−2, S+2/R−1, S+3/R−4. In (B), (C) and (D), the sub-models were superposed to the S0/R0 sub-model based on heavy atom alignment of the first base pair of SLV (residues 689 and 707 in S0/R0). SLI and SLV are represented in deep blue and orange, respectively. Helical axes are represented as red rods and the phosphorus atom at the cleavage site is shown as a sphere. The phosphorus atoms are color-coded according to the catalytic efficiency of the sub-models {[(kcat/KM)/(kcat/KM) ≥ 0.25]: deep orange; [0.25 > (kcat/KM)/(kcat/KM) > 0.01]: light orange and [(kcat/KM)/(kcat/KM) ≤ 0.01]: white}.
© Copyright Policy - creative-commons
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Show All Figures
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gkr1018-F5: Model of an SLI/SLV complex compatible the kinetic data (common-core group 13). (A) Stick representation of the S0/R0 sub-model. (B) Cartoon representation of a superposition of the five variant S/R0 sub-models on the S0/R0 sub-model. The black arrow points at the scissile phosphate of S0/R0. The scissile phosphates of the following substrates are shown from bottom to top: S−1, S0, S+1, S+2, S+3, S+4. (C) Top-down and (D) front views of the superposition of the most catalytically efficient sub-models of each ribozyme. The following S/R pairs are shown from left to right: S−1/R+1, S0/R0, S+1/R−1, S+1/R−2, S+2/R−1, S+3/R−4. In (B), (C) and (D), the sub-models were superposed to the S0/R0 sub-model based on heavy atom alignment of the first base pair of SLV (residues 689 and 707 in S0/R0). SLI and SLV are represented in deep blue and orange, respectively. Helical axes are represented as red rods and the phosphorus atom at the cleavage site is shown as a sphere. The phosphorus atoms are color-coded according to the catalytic efficiency of the sub-models {[(kcat/KM)/(kcat/KM) ≥ 0.25]: deep orange; [0.25 > (kcat/KM)/(kcat/KM) > 0.01]: light orange and [(kcat/KM)/(kcat/KM) ≤ 0.01]: white}.
Mentions: The 3D structure of the S0/R0 sub-model from the selected group reveals that, although the helical axes of stems Ib and V are more or less colinear, the two stems are not coaxial (Figure 5A). Rather, there is a significant perpendicular displacement (dI−V ~ 20 Å) between these axes that results from the structure of the kissing-loop junction. The spatial relation between these two axes is reflected in the structure of variant S/R pairs, as illustrated in Figure 5B–D. The superposition of all S/R pairs containing R0 shows that the scissile phosphates of the high-activity substrates (deep orange spheres in Figure 5B) cover a ~7.5 Å distance on one side of stem Ib, whereas those of low-activity substrates (white spheres in Figure 5B) rotate around the helical axis towards a different side of the helix. Thus, this superposition indicates that limited helical displacements of the scissile phosphate are compatible with productive docking of the substrate internal loop with the catalytic domain.Figure 5.

Bottom Line: Several active substrate/ribozyme pairs were identified, indicating the presence of limited substrate promiscuity for stem Ib variants and helix-length compensation between stems Ib and V. 3D models of the I/V interaction were generated that are compatible with the kinetic data.These models further illustrate the adaptability of the VS ribozyme architecture for substrate cleavage and provide global structural information on the I/V kissing-loop interaction.By exploring higher-order compensatory mutations in RNA our approach brings a deeper understanding of the adaptability of RNA structure, while opening new avenues for RNA research.

View Article: PubMed Central - PubMed

Affiliation: Département de Biochimie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC, Canada.

ABSTRACT
Compensatory mutations in RNA are generally regarded as those that maintain base pairing, and their identification forms the basis of phylogenetic predictions of RNA secondary structure. However, other types of compensatory mutations can provide higher-order structural and evolutionary information. Here, we present a helix-length compensation study for investigating structure-function relationships in RNA. The approach is demonstrated for stem-loop I and stem-loop V of the Neurospora VS ribozyme, which form a kissing-loop interaction important for substrate recognition. To rapidly characterize the substrate specificity (k(cat)/K(M)) of several substrate/ribozyme pairs, a procedure was established for simultaneous kinetic characterization of multiple substrates. Several active substrate/ribozyme pairs were identified, indicating the presence of limited substrate promiscuity for stem Ib variants and helix-length compensation between stems Ib and V. 3D models of the I/V interaction were generated that are compatible with the kinetic data. These models further illustrate the adaptability of the VS ribozyme architecture for substrate cleavage and provide global structural information on the I/V kissing-loop interaction. By exploring higher-order compensatory mutations in RNA our approach brings a deeper understanding of the adaptability of RNA structure, while opening new avenues for RNA research.

Show MeSH