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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.


Experiments testing the formation of duplex P4.(A) Proposed secondary structure for ribozyme TPR1e showing proposed paired regions labeled P1-P5. All duplexes with exception of P4 were identified previously [16]. Nucleotides found accessible by Shape probing are colored in red, while protected nucleotides are shown in blue. C-A pairs are shown with a dot. The numbering scheme corresponds to the cis-acting ribozyme (Fig 1). Note that the duplexes P1 and P2 are formed in trans because the experiments were performed using the trans reaction. Base pairs for duplex P1 are shown in grey because for TPR1e, nucleotides 78–80 appeared accessible by Shape probing. (B) Apparent triphosphorylation rates of the mutated ribozymes in the base covariation experiment of twelve bases in the proposed P4 duplex. The six column graphs are in the same order as the proposed based pairs in P4 of Fig 3A. Each graph shows the triphosphorylation rate of TPR1e on the left, the two single mutants in the middle, and the double mutant on the right. Error bars are standard deviations of triplicate experiments. (C) Shape probing experiments for nucleotides 17 to 46 of the ribozymes TPR1 (empty circles) and TPR1e (colored circles). The normalized Shape signal is shown as a function of nucleotide position. A threshold value (grey, dashed line) is used to distinguish between low Shape reactivity (blue) and high Shape reactivity (red). The colors match the color-coding in Fig 3A. The position of duplexes P1-P5 in TPR1e is indicated by thick, solid, grey, horizontal lines. (D) As in (C) but for nucleotides 57 to 94 in the ribozymes.
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pone.0142559.g003: Experiments testing the formation of duplex P4.(A) Proposed secondary structure for ribozyme TPR1e showing proposed paired regions labeled P1-P5. All duplexes with exception of P4 were identified previously [16]. Nucleotides found accessible by Shape probing are colored in red, while protected nucleotides are shown in blue. C-A pairs are shown with a dot. The numbering scheme corresponds to the cis-acting ribozyme (Fig 1). Note that the duplexes P1 and P2 are formed in trans because the experiments were performed using the trans reaction. Base pairs for duplex P1 are shown in grey because for TPR1e, nucleotides 78–80 appeared accessible by Shape probing. (B) Apparent triphosphorylation rates of the mutated ribozymes in the base covariation experiment of twelve bases in the proposed P4 duplex. The six column graphs are in the same order as the proposed based pairs in P4 of Fig 3A. Each graph shows the triphosphorylation rate of TPR1e on the left, the two single mutants in the middle, and the double mutant on the right. Error bars are standard deviations of triplicate experiments. (C) Shape probing experiments for nucleotides 17 to 46 of the ribozymes TPR1 (empty circles) and TPR1e (colored circles). The normalized Shape signal is shown as a function of nucleotide position. A threshold value (grey, dashed line) is used to distinguish between low Shape reactivity (blue) and high Shape reactivity (red). The colors match the color-coding in Fig 3A. The position of duplexes P1-P5 in TPR1e is indicated by thick, solid, grey, horizontal lines. (D) As in (C) but for nucleotides 57 to 94 in the ribozymes.

Mentions: The mutations selected in TPR1e led to the complementarity between eight bases in nucleotides 31–39 and eight bases in nucleotides 84–92, interrupted only by a single C:A pair (Fig 3A). This suggested the existence of a previously unrecognized duplex, P4. To test whether this duplex formed, base covariation experiments were conducted at six positions in the expected duplex, testing for base pairs G33/C90, U34/A89, G35/C88, C36/G87, U38/A85, and A39/U84 (Fig 3B). The drop in activity of individual mutations G35C and C88G was rescued by the double-mutation suggesting that this portion of the P4 duplex is formed in the active ribozyme. No changes in activity were observed with the mutations of G33C and C90G and with mutations U34A and A89U, suggesting that these bases were not involved in the duplex or that their contribution to duplex stability was not necessary for full activity of the ribozyme. Double mutations at the positions 36/87, 38/85, and 39/84 did not rescue the inhibitory effects of the single mutations, suggesting that the identity of these bases was important for activity. In summary, the base covariation experiments of the proposed P4 duplex were inconclusive because only one out of six positions showed the characteristic deleterious effects of single mutations coupled to a rescue effect of the double mutation.


A Faster Triphosphorylation Ribozyme.

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

Experiments testing the formation of duplex P4.(A) Proposed secondary structure for ribozyme TPR1e showing proposed paired regions labeled P1-P5. All duplexes with exception of P4 were identified previously [16]. Nucleotides found accessible by Shape probing are colored in red, while protected nucleotides are shown in blue. C-A pairs are shown with a dot. The numbering scheme corresponds to the cis-acting ribozyme (Fig 1). Note that the duplexes P1 and P2 are formed in trans because the experiments were performed using the trans reaction. Base pairs for duplex P1 are shown in grey because for TPR1e, nucleotides 78–80 appeared accessible by Shape probing. (B) Apparent triphosphorylation rates of the mutated ribozymes in the base covariation experiment of twelve bases in the proposed P4 duplex. The six column graphs are in the same order as the proposed based pairs in P4 of Fig 3A. Each graph shows the triphosphorylation rate of TPR1e on the left, the two single mutants in the middle, and the double mutant on the right. Error bars are standard deviations of triplicate experiments. (C) Shape probing experiments for nucleotides 17 to 46 of the ribozymes TPR1 (empty circles) and TPR1e (colored circles). The normalized Shape signal is shown as a function of nucleotide position. A threshold value (grey, dashed line) is used to distinguish between low Shape reactivity (blue) and high Shape reactivity (red). The colors match the color-coding in Fig 3A. The position of duplexes P1-P5 in TPR1e is indicated by thick, solid, grey, horizontal lines. (D) As in (C) but for nucleotides 57 to 94 in the ribozymes.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142559.g003: Experiments testing the formation of duplex P4.(A) Proposed secondary structure for ribozyme TPR1e showing proposed paired regions labeled P1-P5. All duplexes with exception of P4 were identified previously [16]. Nucleotides found accessible by Shape probing are colored in red, while protected nucleotides are shown in blue. C-A pairs are shown with a dot. The numbering scheme corresponds to the cis-acting ribozyme (Fig 1). Note that the duplexes P1 and P2 are formed in trans because the experiments were performed using the trans reaction. Base pairs for duplex P1 are shown in grey because for TPR1e, nucleotides 78–80 appeared accessible by Shape probing. (B) Apparent triphosphorylation rates of the mutated ribozymes in the base covariation experiment of twelve bases in the proposed P4 duplex. The six column graphs are in the same order as the proposed based pairs in P4 of Fig 3A. Each graph shows the triphosphorylation rate of TPR1e on the left, the two single mutants in the middle, and the double mutant on the right. Error bars are standard deviations of triplicate experiments. (C) Shape probing experiments for nucleotides 17 to 46 of the ribozymes TPR1 (empty circles) and TPR1e (colored circles). The normalized Shape signal is shown as a function of nucleotide position. A threshold value (grey, dashed line) is used to distinguish between low Shape reactivity (blue) and high Shape reactivity (red). The colors match the color-coding in Fig 3A. The position of duplexes P1-P5 in TPR1e is indicated by thick, solid, grey, horizontal lines. (D) As in (C) but for nucleotides 57 to 94 in the ribozymes.
Mentions: The mutations selected in TPR1e led to the complementarity between eight bases in nucleotides 31–39 and eight bases in nucleotides 84–92, interrupted only by a single C:A pair (Fig 3A). This suggested the existence of a previously unrecognized duplex, P4. To test whether this duplex formed, base covariation experiments were conducted at six positions in the expected duplex, testing for base pairs G33/C90, U34/A89, G35/C88, C36/G87, U38/A85, and A39/U84 (Fig 3B). The drop in activity of individual mutations G35C and C88G was rescued by the double-mutation suggesting that this portion of the P4 duplex is formed in the active ribozyme. No changes in activity were observed with the mutations of G33C and C90G and with mutations U34A and A89U, suggesting that these bases were not involved in the duplex or that their contribution to duplex stability was not necessary for full activity of the ribozyme. Double mutations at the positions 36/87, 38/85, and 39/84 did not rescue the inhibitory effects of the single mutations, suggesting that the identity of these bases was important for activity. In summary, the base covariation experiments of the proposed P4 duplex were inconclusive because only one out of six positions showed the characteristic deleterious effects of single mutations coupled to a rescue effect of the double mutation.

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.