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A Faster Triphosphorylation Ribozyme.

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

Bottom Line: One ribozyme (TPR1) was analyzed in more detail.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.

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

Determination of optimal TPR1e triphosphorylation conditions.(A) Observed triphosphorylation rate as function of the temperature, at 50 mM Tmp, 100 mM MgCl2, and 50 mM Tris/HCl pH 8.3 (B) Influence of the trimetaphosphate concentration on the observed reaction kinetics at 40°C, and with an excess of 400 mM MgCl2 over Tmp. (C) Influence of the free Mg2+ concentration on the triphosphorylation rate at 40°C and 150 mM Tmp. The free Mg2+ concentration is the total Mg2+ concentration minus the concentration of Tmp because each Tmp appears to be coordinated by one Mg2+ at these concentrations and pH 8.3 [16]. The grey arrows indicate the optimum condition for each series of experiments. Note that the scale in (A) is different from the scale in (B) and (C). Error bars are standard deviations of triplicate experiments.
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pone.0142559.g004: Determination of optimal TPR1e triphosphorylation conditions.(A) Observed triphosphorylation rate as function of the temperature, at 50 mM Tmp, 100 mM MgCl2, and 50 mM Tris/HCl pH 8.3 (B) Influence of the trimetaphosphate concentration on the observed reaction kinetics at 40°C, and with an excess of 400 mM MgCl2 over Tmp. (C) Influence of the free Mg2+ concentration on the triphosphorylation rate at 40°C and 150 mM Tmp. The free Mg2+ concentration is the total Mg2+ concentration minus the concentration of Tmp because each Tmp appears to be coordinated by one Mg2+ at these concentrations and pH 8.3 [16]. The grey arrows indicate the optimum condition for each series of experiments. Note that the scale in (A) is different from the scale in (B) and (C). Error bars are standard deviations of triplicate experiments.

Mentions: To identify the optimal reaction conditions for the evolved ribozyme (TPR1e), the influence of temperature, Tmp concentration, and Mg2+ concentration were analyzed sequentially. First, the temperature was varied between 5°C and 60°C, and an optimum at 40°C was determined (Fig 4A). At 40°C, the optimal Tmp concentration was at 150 mM (Fig 4B), and the optimal concentration of free Mg2+ at 150mM Tmp was at 500 mM (Fig 4C). Interestingly, the dependence on the Tmp concentration at these high Mg2+ conditions and high temperature showed a cooperative effect, with a significantly higher rate at 100 mM Tmp (5.7 min-1) than what would result from doubling the rate at 50 mM Tmp (2 • 0.96 min-1 = 1.92 min-1). This suggested that a second molecule of trimetaphosphate binds to the ribozyme and/or the first trimetaphosphate molecule and increases the reaction rate. This effect was not observed with the parent ribozyme TPR1 [16]. The mechanism of the cooperative effect is currently unclear. Notably, the effect did not appear at more modest Mg2+ concentrations (~50 mM free Mg2+ versus 400 mM free Mg2+) and lower temperature (22°C vs. 40°C), where a linear correlation between Tmp concentration and reaction rate was observed (Fig 5 and [16]). At the optimal conditions (40°C, 150 mM Tmp, 650 mM total MgCl2, 50 mM Tris/HCl pH 8.3) the cooperative effect afforded a reaction rate of 6.8 min-1.


A Faster Triphosphorylation Ribozyme.

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

Determination of optimal TPR1e triphosphorylation conditions.(A) Observed triphosphorylation rate as function of the temperature, at 50 mM Tmp, 100 mM MgCl2, and 50 mM Tris/HCl pH 8.3 (B) Influence of the trimetaphosphate concentration on the observed reaction kinetics at 40°C, and with an excess of 400 mM MgCl2 over Tmp. (C) Influence of the free Mg2+ concentration on the triphosphorylation rate at 40°C and 150 mM Tmp. The free Mg2+ concentration is the total Mg2+ concentration minus the concentration of Tmp because each Tmp appears to be coordinated by one Mg2+ at these concentrations and pH 8.3 [16]. The grey arrows indicate the optimum condition for each series of experiments. Note that the scale in (A) is different from the scale in (B) and (C). Error bars are standard deviations of triplicate experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142559.g004: Determination of optimal TPR1e triphosphorylation conditions.(A) Observed triphosphorylation rate as function of the temperature, at 50 mM Tmp, 100 mM MgCl2, and 50 mM Tris/HCl pH 8.3 (B) Influence of the trimetaphosphate concentration on the observed reaction kinetics at 40°C, and with an excess of 400 mM MgCl2 over Tmp. (C) Influence of the free Mg2+ concentration on the triphosphorylation rate at 40°C and 150 mM Tmp. The free Mg2+ concentration is the total Mg2+ concentration minus the concentration of Tmp because each Tmp appears to be coordinated by one Mg2+ at these concentrations and pH 8.3 [16]. The grey arrows indicate the optimum condition for each series of experiments. Note that the scale in (A) is different from the scale in (B) and (C). Error bars are standard deviations of triplicate experiments.
Mentions: To identify the optimal reaction conditions for the evolved ribozyme (TPR1e), the influence of temperature, Tmp concentration, and Mg2+ concentration were analyzed sequentially. First, the temperature was varied between 5°C and 60°C, and an optimum at 40°C was determined (Fig 4A). At 40°C, the optimal Tmp concentration was at 150 mM (Fig 4B), and the optimal concentration of free Mg2+ at 150mM Tmp was at 500 mM (Fig 4C). Interestingly, the dependence on the Tmp concentration at these high Mg2+ conditions and high temperature showed a cooperative effect, with a significantly higher rate at 100 mM Tmp (5.7 min-1) than what would result from doubling the rate at 50 mM Tmp (2 • 0.96 min-1 = 1.92 min-1). This suggested that a second molecule of trimetaphosphate binds to the ribozyme and/or the first trimetaphosphate molecule and increases the reaction rate. This effect was not observed with the parent ribozyme TPR1 [16]. The mechanism of the cooperative effect is currently unclear. Notably, the effect did not appear at more modest Mg2+ concentrations (~50 mM free Mg2+ versus 400 mM free Mg2+) and lower temperature (22°C vs. 40°C), where a linear correlation between Tmp concentration and reaction rate was observed (Fig 5 and [16]). At the optimal conditions (40°C, 150 mM Tmp, 650 mM total MgCl2, 50 mM Tris/HCl pH 8.3) the cooperative effect afforded a reaction rate of 6.8 min-1.

Bottom Line: One ribozyme (TPR1) was analyzed in more detail.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.

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