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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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Single-substrate kinetic analysis of R0. (A) Cleavage of 5′-32P-labeled S0 by R0 (at 37.5 nM) monitored by denaturing gel electrophoresis. (B) The percentage of remaining substrate was plotted against time. The data were fitted to the non-linear equation F = Ae−(kobs *t)+ F∞, to extract the value of kobs (kobs = 0.332 ± 0.008 min−1; R2 = 1.00). (C) Cleavage reactions were performed at various concentrations of R0, and the value of kobs was plotted against ribozyme concentration. For all kobs values, error bars are smaller than the data point on the graph. The data were fitted to a single exponential equation, and the kcat/KM is extracted by linear regression (kcat/KM = value of the slope = 11.2 min−1µM−1 and R2 = 0.99).
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gkr1018-F2: Single-substrate kinetic analysis of R0. (A) Cleavage of 5′-32P-labeled S0 by R0 (at 37.5 nM) monitored by denaturing gel electrophoresis. (B) The percentage of remaining substrate was plotted against time. The data were fitted to the non-linear equation F = Ae−(kobs *t)+ F∞, to extract the value of kobs (kobs = 0.332 ± 0.008 min−1; R2 = 1.00). (C) Cleavage reactions were performed at various concentrations of R0, and the value of kobs was plotted against ribozyme concentration. For all kobs values, error bars are smaller than the data point on the graph. The data were fitted to a single exponential equation, and the kcat/KM is extracted by linear regression (kcat/KM = value of the slope = 11.2 min−1µM−1 and R2 = 0.99).

Mentions: We first examined the specificity of R0 for each substrate individually by determining the second-order rate constant (kcat/KM) under single-turnover conditions (18), as illustrated for S0 in Figure 2. Cleavage reactions were performed with 32P-labeled S0 and excess R0 and monitored by denaturing gel electrophoresis (Figure 2A). The percentage of remaining substrate was plotted against time to derive the value of the first-order rate constant kobs (Figure 2B). For S−1 bp, S0 and S+1bp a linear relationship is observed when kobs is plotted as a function of R0 concentration, and the values of catalytic efficiency (kcat/KM) were obtained from the slope of this graph (Figure 2C and Table 1). For all other substrates (S−2 bp, S+2 bp, S+3bp and S+4 bp), the value of kobs is below the detection limit (≤ 0.001 min−1) at R0 concentrations up to 5 µM, and therefore the value of kcat/KM cannot be determined (Table 1). To insure that the lack of reactivity of certain substrates is not due to major folding defects, they were analyzed by native gel electrophoresis (Supplementary Figure S1). All substrates, with exception of S−2 bp, migrate as a single band on the gel, at a position typical of small RNA hairpins, in agreement with the predicted secondary structure (38). Most of the S−2bp substrate migrates slower, indicating that it adopts a duplex conformation that is incompatible with cleavage by R0.Figure 2.


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)

Single-substrate kinetic analysis of R0. (A) Cleavage of 5′-32P-labeled S0 by R0 (at 37.5 nM) monitored by denaturing gel electrophoresis. (B) The percentage of remaining substrate was plotted against time. The data were fitted to the non-linear equation F = Ae−(kobs *t)+ F∞, to extract the value of kobs (kobs = 0.332 ± 0.008 min−1; R2 = 1.00). (C) Cleavage reactions were performed at various concentrations of R0, and the value of kobs was plotted against ribozyme concentration. For all kobs values, error bars are smaller than the data point on the graph. The data were fitted to a single exponential equation, and the kcat/KM is extracted by linear regression (kcat/KM = value of the slope = 11.2 min−1µM−1 and R2 = 0.99).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3299992&req=5

gkr1018-F2: Single-substrate kinetic analysis of R0. (A) Cleavage of 5′-32P-labeled S0 by R0 (at 37.5 nM) monitored by denaturing gel electrophoresis. (B) The percentage of remaining substrate was plotted against time. The data were fitted to the non-linear equation F = Ae−(kobs *t)+ F∞, to extract the value of kobs (kobs = 0.332 ± 0.008 min−1; R2 = 1.00). (C) Cleavage reactions were performed at various concentrations of R0, and the value of kobs was plotted against ribozyme concentration. For all kobs values, error bars are smaller than the data point on the graph. The data were fitted to a single exponential equation, and the kcat/KM is extracted by linear regression (kcat/KM = value of the slope = 11.2 min−1µM−1 and R2 = 0.99).
Mentions: We first examined the specificity of R0 for each substrate individually by determining the second-order rate constant (kcat/KM) under single-turnover conditions (18), as illustrated for S0 in Figure 2. Cleavage reactions were performed with 32P-labeled S0 and excess R0 and monitored by denaturing gel electrophoresis (Figure 2A). The percentage of remaining substrate was plotted against time to derive the value of the first-order rate constant kobs (Figure 2B). For S−1 bp, S0 and S+1bp a linear relationship is observed when kobs is plotted as a function of R0 concentration, and the values of catalytic efficiency (kcat/KM) were obtained from the slope of this graph (Figure 2C and Table 1). For all other substrates (S−2 bp, S+2 bp, S+3bp and S+4 bp), the value of kobs is below the detection limit (≤ 0.001 min−1) at R0 concentrations up to 5 µM, and therefore the value of kcat/KM cannot be determined (Table 1). To insure that the lack of reactivity of certain substrates is not due to major folding defects, they were analyzed by native gel electrophoresis (Supplementary Figure S1). All substrates, with exception of S−2 bp, migrate as a single band on the gel, at a position typical of small RNA hairpins, in agreement with the predicted secondary structure (38). Most of the S−2bp substrate migrates slower, indicating that it adopts a duplex conformation that is incompatible with cleavage by R0.Figure 2.

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
Related in: MedlinePlus