Limits...
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.

Show MeSH
Simultaneous kinetic characterization of R−3bp with multiple substrates. The cleavage reactions of 5′-32P-labeled substrates (S) by R−3bp (at 100 nM) were monitored by denaturing gel electrophoresis. A DNA hairpin was used as a normalization control (C).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3299992&req=5

gkr1018-F3: Simultaneous kinetic characterization of R−3bp with multiple substrates. The cleavage reactions of 5′-32P-labeled substrates (S) by R−3bp (at 100 nM) were monitored by denaturing gel electrophoresis. A DNA hairpin was used as a normalization control (C).

Mentions: Single-substrate kinetic studies performed with R0 are fairly tedious and time-consuming, especially given that several time-courses are needed to derive kcat/KM values for an individual substrate, each kcat/KM are determined at least three times, and multiple substrates are being investigated. To accelerate the kinetic characterization of the remaining S/R variant pairs (Figure 1B), we set up a multiple-substrate kinetic procedure in which one enzyme is simultaneously incubated with a mixture of substrates (29). Single-turnover conditions were chosen for our studies because the enzyme is in excess with respect to all substrates, and there should be no competition between the various substrates. Given the different sizes of substrates investigated (Figure 1B), the disappearance of any given substrate is easily monitored by denaturing gel electrophoresis (Figure 3). The percentage of remaining substrate can be reliably estimated from the ratio of substrate remaining at time t to that at time zero provided that normalization of input RNA in each lane can be achieved. For normalization, we initially added a non-cleavable DNA control in the cleavage reaction, but later found that quantification is more reliable using the non-cleavable S−2bp RNA that gives a better-defined band on the gel (Figure 3).Figure 3.


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)

Simultaneous kinetic characterization of R−3bp with multiple substrates. The cleavage reactions of 5′-32P-labeled substrates (S) by R−3bp (at 100 nM) were monitored by denaturing gel electrophoresis. A DNA hairpin was used as a normalization control (C).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1018-F3: Simultaneous kinetic characterization of R−3bp with multiple substrates. The cleavage reactions of 5′-32P-labeled substrates (S) by R−3bp (at 100 nM) were monitored by denaturing gel electrophoresis. A DNA hairpin was used as a normalization control (C).
Mentions: Single-substrate kinetic studies performed with R0 are fairly tedious and time-consuming, especially given that several time-courses are needed to derive kcat/KM values for an individual substrate, each kcat/KM are determined at least three times, and multiple substrates are being investigated. To accelerate the kinetic characterization of the remaining S/R variant pairs (Figure 1B), we set up a multiple-substrate kinetic procedure in which one enzyme is simultaneously incubated with a mixture of substrates (29). Single-turnover conditions were chosen for our studies because the enzyme is in excess with respect to all substrates, and there should be no competition between the various substrates. Given the different sizes of substrates investigated (Figure 1B), the disappearance of any given substrate is easily monitored by denaturing gel electrophoresis (Figure 3). The percentage of remaining substrate can be reliably estimated from the ratio of substrate remaining at time t to that at time zero provided that normalization of input RNA in each lane can be achieved. For normalization, we initially added a non-cleavable DNA control in the cleavage reaction, but later found that quantification is more reliable using the non-cleavable S−2bp RNA that gives a better-defined band on the gel (Figure 3).Figure 3.

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