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A Faster Triphosphorylation Ribozyme.

Dolan GF, Akoopie A, Müller UF - PLoS ONE (2015)

Bottom Line: To identify a triphosphorylation ribozyme that catalyzes faster triphosphorylation, and possibly learn about its secondary structure TPR1 was subjected to a doped selection.The resulting ribozyme, TPR1e, contains seven mutations relative to TPR1, displays a previously unidentified duplex that constrains the ribozyme's structure, and reacts at a 24-fold faster rate than the parent ribozyme.Under optimal conditions (150 mM Tmp, 650 mM MgCl2, 40°C), the triphosphorylation rate of TRP1e reaches 6.8 min-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
In support of the RNA world hypothesis, previous studies identified trimetaphosphate (Tmp) as a plausible energy source for RNA world organisms. In one of these studies, catalytic RNAs (ribozymes) that catalyze the triphosphorylation of RNA 5'-hydroxyl groups using Tmp were obtained by in vitro selection. One ribozyme (TPR1) was analyzed in more detail. TPR1 catalyzes the triphosphorylation reaction to a rate of 0.013 min-1 under selection conditions (50 mM Tmp, 100 mM MgCl2, 22°C). To identify a triphosphorylation ribozyme that catalyzes faster triphosphorylation, and possibly learn about its secondary structure TPR1 was subjected to a doped selection. The resulting ribozyme, TPR1e, contains seven mutations relative to TPR1, displays a previously unidentified duplex that constrains the ribozyme's structure, and reacts at a 24-fold faster rate than the parent ribozyme. Under optimal conditions (150 mM Tmp, 650 mM MgCl2, 40°C), the triphosphorylation rate of TRP1e reaches 6.8 min-1.

No MeSH data available.


Related in: MedlinePlus

Triphosphorylation kinetics of central ribozyme variants in this study.The starting point of the doped selection was the ribozyme TPR1 (empty circles). It has a kobs of 0.013 min-1 under selection conditions (100 mM MgCl2, 50 mM trimetaphosphate, 50 mM Tris/HCl pH 8.3). The most efficient isolate from the doped selection was a 16-mutation variant called clone 11 (filled triangles, kobs of 0.21 min-1). After removal of unnecessary mutations a 5-mutation variant called TPR1-II resulted (open squares, kobs of 0.25 min-1). Two mutations that arose independently were introduced to yield TPR1e (filled squares), a 7-mutation variant with a kobs of 0.31 min-1. Lines are single-exponential curve fits to the data. Error bars denote the standard deviations from triplicate experiments.
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pone.0142559.g002: Triphosphorylation kinetics of central ribozyme variants in this study.The starting point of the doped selection was the ribozyme TPR1 (empty circles). It has a kobs of 0.013 min-1 under selection conditions (100 mM MgCl2, 50 mM trimetaphosphate, 50 mM Tris/HCl pH 8.3). The most efficient isolate from the doped selection was a 16-mutation variant called clone 11 (filled triangles, kobs of 0.21 min-1). After removal of unnecessary mutations a 5-mutation variant called TPR1-II resulted (open squares, kobs of 0.25 min-1). Two mutations that arose independently were introduced to yield TPR1e (filled squares), a 7-mutation variant with a kobs of 0.31 min-1. Lines are single-exponential curve fits to the data. Error bars denote the standard deviations from triplicate experiments.

Mentions: Of the eighty-three sequences assayed for triphosphorylation kinetics, fifty-eight displayed triphosphorylation kinetics at least as fast as the parent ribozyme TPR1. The six fastest ribozyme clones contained the same set of two mutations (G37C, A38U), and five of them contained the mutation C86A. The fastest ribozyme was clone 11, with 16 mutations relative to TPR1 and a self-triphosphorylation rate of 0.21 ± 0.02 min-1 under selection conditions (50 mM Tmp, 100 mM MgCl2, 50 mM Tris/HCl pH 8.3) (Fig 2). This ribozyme was isolated from the selection line with decreased Tmp concentration. To identify the mutations in clone 11 that were necessary for improved triphosphorylation kinetics, all 16 mutations were individually reverted to the parent sequence (S2 Fig). Only five of the sixteen mutations were necessary for increased activity (U28C, G37C, A38U, C86A, A90C). This ribozyme was called TPR1_II. The reduction to five mutations was possible by the finding (see materials and methods) that four of the eleven nonessential mutations appeared to elongate and stabilize the long central P5 duplex of the ribozyme (U55C, A56U, U58C, U64G). These elongating mutations were only necessary in the context of two destabilizing mutations within the P5 duplex of clone 11 (A51U, C52G). All clone 11 mutations within the P5 stem-loop could be deleted while maintaining (and even increasing) catalytic activity. TPR1_II showed a self-triphosphorylation rate of 0.25 ± 0.03 min-1 under selection conditions (Fig 2 and S2 Fig).


A Faster Triphosphorylation Ribozyme.

Dolan GF, Akoopie A, Müller UF - PLoS ONE (2015)

Triphosphorylation kinetics of central ribozyme variants in this study.The starting point of the doped selection was the ribozyme TPR1 (empty circles). It has a kobs of 0.013 min-1 under selection conditions (100 mM MgCl2, 50 mM trimetaphosphate, 50 mM Tris/HCl pH 8.3). The most efficient isolate from the doped selection was a 16-mutation variant called clone 11 (filled triangles, kobs of 0.21 min-1). After removal of unnecessary mutations a 5-mutation variant called TPR1-II resulted (open squares, kobs of 0.25 min-1). Two mutations that arose independently were introduced to yield TPR1e (filled squares), a 7-mutation variant with a kobs of 0.31 min-1. Lines are single-exponential curve fits to the data. Error bars denote the standard deviations from triplicate experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4636267&req=5

pone.0142559.g002: Triphosphorylation kinetics of central ribozyme variants in this study.The starting point of the doped selection was the ribozyme TPR1 (empty circles). It has a kobs of 0.013 min-1 under selection conditions (100 mM MgCl2, 50 mM trimetaphosphate, 50 mM Tris/HCl pH 8.3). The most efficient isolate from the doped selection was a 16-mutation variant called clone 11 (filled triangles, kobs of 0.21 min-1). After removal of unnecessary mutations a 5-mutation variant called TPR1-II resulted (open squares, kobs of 0.25 min-1). Two mutations that arose independently were introduced to yield TPR1e (filled squares), a 7-mutation variant with a kobs of 0.31 min-1. Lines are single-exponential curve fits to the data. Error bars denote the standard deviations from triplicate experiments.
Mentions: Of the eighty-three sequences assayed for triphosphorylation kinetics, fifty-eight displayed triphosphorylation kinetics at least as fast as the parent ribozyme TPR1. The six fastest ribozyme clones contained the same set of two mutations (G37C, A38U), and five of them contained the mutation C86A. The fastest ribozyme was clone 11, with 16 mutations relative to TPR1 and a self-triphosphorylation rate of 0.21 ± 0.02 min-1 under selection conditions (50 mM Tmp, 100 mM MgCl2, 50 mM Tris/HCl pH 8.3) (Fig 2). This ribozyme was isolated from the selection line with decreased Tmp concentration. To identify the mutations in clone 11 that were necessary for improved triphosphorylation kinetics, all 16 mutations were individually reverted to the parent sequence (S2 Fig). Only five of the sixteen mutations were necessary for increased activity (U28C, G37C, A38U, C86A, A90C). This ribozyme was called TPR1_II. The reduction to five mutations was possible by the finding (see materials and methods) that four of the eleven nonessential mutations appeared to elongate and stabilize the long central P5 duplex of the ribozyme (U55C, A56U, U58C, U64G). These elongating mutations were only necessary in the context of two destabilizing mutations within the P5 duplex of clone 11 (A51U, C52G). All clone 11 mutations within the P5 stem-loop could be deleted while maintaining (and even increasing) catalytic activity. TPR1_II showed a self-triphosphorylation rate of 0.25 ± 0.03 min-1 under selection conditions (Fig 2 and S2 Fig).

Bottom Line: To identify a triphosphorylation ribozyme that catalyzes faster triphosphorylation, and possibly learn about its secondary structure TPR1 was subjected to a doped selection.The resulting ribozyme, TPR1e, contains seven mutations relative to TPR1, displays a previously unidentified duplex that constrains the ribozyme's structure, and reacts at a 24-fold faster rate than the parent ribozyme.Under optimal conditions (150 mM Tmp, 650 mM MgCl2, 40°C), the triphosphorylation rate of TRP1e reaches 6.8 min-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
In support of the RNA world hypothesis, previous studies identified trimetaphosphate (Tmp) as a plausible energy source for RNA world organisms. In one of these studies, catalytic RNAs (ribozymes) that catalyze the triphosphorylation of RNA 5'-hydroxyl groups using Tmp were obtained by in vitro selection. One ribozyme (TPR1) was analyzed in more detail. TPR1 catalyzes the triphosphorylation reaction to a rate of 0.013 min-1 under selection conditions (50 mM Tmp, 100 mM MgCl2, 22°C). To identify a triphosphorylation ribozyme that catalyzes faster triphosphorylation, and possibly learn about its secondary structure TPR1 was subjected to a doped selection. The resulting ribozyme, TPR1e, contains seven mutations relative to TPR1, displays a previously unidentified duplex that constrains the ribozyme's structure, and reacts at a 24-fold faster rate than the parent ribozyme. Under optimal conditions (150 mM Tmp, 650 mM MgCl2, 40°C), the triphosphorylation rate of TRP1e reaches 6.8 min-1.

No MeSH data available.


Related in: MedlinePlus