4 research outputs found

    Triphosphorylation kinetics of TPR1e.

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    <p><b>(A)</b> At a Tmp concentration of 1 mM, the triphosphorylation kinetics are shown for synthetic seawater at 22°C (black triangles, k<sub>obs</sub> = 0.0014 min<sup>-1</sup>, max = 93%), and at 40°C (empty squares, k<sub>obs</sub> = 0.0039 min<sup>-1</sup>, max = 97%). For comparison, the reaction kinetics are shown for 54 mM MgCl<sub>2</sub> in Tris/HCl pH 8.3 at 22°C (black circles, k<sub>obs</sub> = 0.020 min<sup>-1</sup>, max = 93%). This latter condition lacks all seawater components with exception of Mg<sup>2+</sup>. Error bars are standard deviations from triplicate experiments, and are smaller than the symbols if not visible. Curves are single-exponential fits to the data. <b>(B)</b> Titration of the Tmp concentration in the reaction at 22°C, in synthetic seawater (black triangles) and in 54 mM MgCl<sub>2</sub> with 50 mM Tris/HCl pH 8.3 (black circles). The offset between the linear fits (grey lines) is 15-fold, on average. <b>(C)</b> Titration of sodium chloride into a triphosphorylation ribozyme reaction containing 50 mM Tmp and 140 mM MgCl<sub>2</sub>. The grey line is a single-exponential fit to the data (with offset) and identifies a 1.9-fold reduction in k<sub>obs</sub> at 470 mM [NaCl], the same NaCl concentration as in synthetic seawater (dashed line). Error bars are standard deviations from triplicate experiments, and are smaller than the symbols if not visible.</p

    Determination of optimal TPR1e triphosphorylation conditions.

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    <p><b>(A)</b> Observed triphosphorylation rate as function of the temperature, at 50 mM Tmp, 100 mM MgCl<sub>2</sub>, and 50 mM Tris/HCl pH 8.3 <b>(B)</b> Influence of the trimetaphosphate concentration on the observed reaction kinetics at 40°C, and with an excess of 400 mM MgCl<sub>2</sub> over Tmp. <b>(C)</b> Influence of the free Mg<sup>2+</sup> concentration on the triphosphorylation rate at 40°C and 150 mM Tmp. The free Mg<sup>2+</sup> concentration is the total Mg<sup>2+</sup> concentration minus the concentration of Tmp because each Tmp appears to be coordinated by one Mg<sup>2+</sup> at these concentrations and pH 8.3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142559#pone.0142559.ref016" target="_blank">16</a>]. 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.</p

    Triphosphorylation kinetics of central ribozyme variants in this study.

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    <p>The starting point of the doped selection was the ribozyme TPR1 (empty circles). It has a k<sub>obs</sub> of 0.013 min<sup>-1</sup> under selection conditions (100 mM MgCl<sub>2</sub>, 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, k<sub>obs</sub> of 0.21 min<sup>-1</sup>). After removal of unnecessary mutations a 5-mutation variant called TPR1-II resulted (open squares, k<sub>obs</sub> of 0.25 min<sup>-1</sup>). Two mutations that arose independently were introduced to yield TPR1e (filled squares), a 7-mutation variant with a k<sub>obs</sub> of 0.31 min<sup>-1</sup>. Lines are single-exponential curve fits to the data. Error bars denote the standard deviations from triplicate experiments.</p