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RNA synthesis by in vitro selected ribozymes for recreating an RNA world.

Martin LL, Unrau PJ, Müller UF - Life (Basel) (2015)

Bottom Line: The RNA world hypothesis states that during an early stage of life, RNA molecules functioned as genome and as the only genome-encoded catalyst.This hypothesis is supported by several lines of evidence, one of which is the in vitro selection of catalytic RNAs (ribozymes) in the laboratory for a wide range of reactions that might have been used by RNA world organisms.These ribozyme classes catalyze nucleoside synthesis, triphosphorylation, and the polymerization of nucleoside triphosphates.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada. lyssam@sfu.ca.

ABSTRACT
The RNA world hypothesis states that during an early stage of life, RNA molecules functioned as genome and as the only genome-encoded catalyst. This hypothesis is supported by several lines of evidence, one of which is the in vitro selection of catalytic RNAs (ribozymes) in the laboratory for a wide range of reactions that might have been used by RNA world organisms. This review focuses on three types of ribozymes that could have been involved in the synthesis of RNA, the core activity in the self-replication of RNA world organisms. These ribozyme classes catalyze nucleoside synthesis, triphosphorylation, and the polymerization of nucleoside triphosphates. The strengths and weaknesses regarding each ribozyme's possible function in a self-replicating RNA network are described, together with the obstacles that need to be overcome before an RNA world organism can be generated in the laboratory.

No MeSH data available.


Related in: MedlinePlus

Reaction and secondary structure of a triphosphorylation ribozyme. (A) Gel shift assay of the triphosphorylation reaction at different reaction times with Tmp (5 min to 3 h). An 8-nucleotide fragment is cleaved from the 5'-terminus of the ribozyme after incubation with Tmp. The short length of this fragment allows separating the fragments with a 5'-terminal hydroxyl group (5'-OH) and with a 5'-triphosphate (5'-PPP). The percent of the fragment that are triphosphorylated are plotted as a function of the incubation with trimetaphosphate, which allows determining the single-exponential reaction rate; (B) Secondary structure of TPR1 resulting from Shape probing and base covariation experiments.
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life-05-00247-f004: Reaction and secondary structure of a triphosphorylation ribozyme. (A) Gel shift assay of the triphosphorylation reaction at different reaction times with Tmp (5 min to 3 h). An 8-nucleotide fragment is cleaved from the 5'-terminus of the ribozyme after incubation with Tmp. The short length of this fragment allows separating the fragments with a 5'-terminal hydroxyl group (5'-OH) and with a 5'-triphosphate (5'-PPP). The percent of the fragment that are triphosphorylated are plotted as a function of the incubation with trimetaphosphate, which allows determining the single-exponential reaction rate; (B) Secondary structure of TPR1 resulting from Shape probing and base covariation experiments.

Mentions: Eight of the selected triphosphorylation ribozymes were analyzed for their reaction kinetics, and had rates between 0.013 min−1 and 0.028 min−1, under selection conditions (50 mM Tmp, 100 mM total MgCl2, pH 8.3) [40]. One of the ribozymes reacted to 83% and was analyzed in more detail. It was truncated from 182 nucleotides to 96 nucleotides while maintaining full activity. This truncated ribozyme was termed TPR1 (triphosphorylation ribozyme 1). Under optimal conditions (100 mM Tmp, 500 mM total MgCl2, pH 8.1) the TPR1-catalyzed reaction rate was 0.16 min−1, about 107-fold faster than the uncatalyzed reaction. The Km for Tmp was 30 mM, leading to an apparent catalytic efficiency of 5.3 M−1·min−1. A modification at its 5'-terminus allowed the reaction to proceed in trans, allowing a 14-nucleotide long RNA oligonucleotide to be triphosphorylated at its 5'-terminus by the ribozyme. This allowed the confirmation of the triphosphorylated product by mass spectroscopy. The dependence of the rate on the Mg2+ concentration suggested that each Tmp molecule coordinated one Mg2+ ion in a bidentate fashion and a second Mg2+ ion with the remaining negatively charged oxygen. The pH dependence of the reaction kinetics showed that a single deprotonation step was rate-limiting for the reaction, presumably the deprotonation of the 5'-hydroxyl group that made the nucleophilic attack on the trimetaphosphate. The secondary structure of the triphosphorylation ribozyme was analyzed using SHAPE analysis [75] and base covariation analysis (Figure 4). The ribozyme appears to be highly compact, with the help of a 4-way helical junction. It is currently unclear how this 4-way helical junction is arranged in three dimensions, and how Tmp is bound by the ribozyme.


RNA synthesis by in vitro selected ribozymes for recreating an RNA world.

Martin LL, Unrau PJ, Müller UF - Life (Basel) (2015)

Reaction and secondary structure of a triphosphorylation ribozyme. (A) Gel shift assay of the triphosphorylation reaction at different reaction times with Tmp (5 min to 3 h). An 8-nucleotide fragment is cleaved from the 5'-terminus of the ribozyme after incubation with Tmp. The short length of this fragment allows separating the fragments with a 5'-terminal hydroxyl group (5'-OH) and with a 5'-triphosphate (5'-PPP). The percent of the fragment that are triphosphorylated are plotted as a function of the incubation with trimetaphosphate, which allows determining the single-exponential reaction rate; (B) Secondary structure of TPR1 resulting from Shape probing and base covariation experiments.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4390851&req=5

life-05-00247-f004: Reaction and secondary structure of a triphosphorylation ribozyme. (A) Gel shift assay of the triphosphorylation reaction at different reaction times with Tmp (5 min to 3 h). An 8-nucleotide fragment is cleaved from the 5'-terminus of the ribozyme after incubation with Tmp. The short length of this fragment allows separating the fragments with a 5'-terminal hydroxyl group (5'-OH) and with a 5'-triphosphate (5'-PPP). The percent of the fragment that are triphosphorylated are plotted as a function of the incubation with trimetaphosphate, which allows determining the single-exponential reaction rate; (B) Secondary structure of TPR1 resulting from Shape probing and base covariation experiments.
Mentions: Eight of the selected triphosphorylation ribozymes were analyzed for their reaction kinetics, and had rates between 0.013 min−1 and 0.028 min−1, under selection conditions (50 mM Tmp, 100 mM total MgCl2, pH 8.3) [40]. One of the ribozymes reacted to 83% and was analyzed in more detail. It was truncated from 182 nucleotides to 96 nucleotides while maintaining full activity. This truncated ribozyme was termed TPR1 (triphosphorylation ribozyme 1). Under optimal conditions (100 mM Tmp, 500 mM total MgCl2, pH 8.1) the TPR1-catalyzed reaction rate was 0.16 min−1, about 107-fold faster than the uncatalyzed reaction. The Km for Tmp was 30 mM, leading to an apparent catalytic efficiency of 5.3 M−1·min−1. A modification at its 5'-terminus allowed the reaction to proceed in trans, allowing a 14-nucleotide long RNA oligonucleotide to be triphosphorylated at its 5'-terminus by the ribozyme. This allowed the confirmation of the triphosphorylated product by mass spectroscopy. The dependence of the rate on the Mg2+ concentration suggested that each Tmp molecule coordinated one Mg2+ ion in a bidentate fashion and a second Mg2+ ion with the remaining negatively charged oxygen. The pH dependence of the reaction kinetics showed that a single deprotonation step was rate-limiting for the reaction, presumably the deprotonation of the 5'-hydroxyl group that made the nucleophilic attack on the trimetaphosphate. The secondary structure of the triphosphorylation ribozyme was analyzed using SHAPE analysis [75] and base covariation analysis (Figure 4). The ribozyme appears to be highly compact, with the help of a 4-way helical junction. It is currently unclear how this 4-way helical junction is arranged in three dimensions, and how Tmp is bound by the ribozyme.

Bottom Line: The RNA world hypothesis states that during an early stage of life, RNA molecules functioned as genome and as the only genome-encoded catalyst.This hypothesis is supported by several lines of evidence, one of which is the in vitro selection of catalytic RNAs (ribozymes) in the laboratory for a wide range of reactions that might have been used by RNA world organisms.These ribozyme classes catalyze nucleoside synthesis, triphosphorylation, and the polymerization of nucleoside triphosphates.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada. lyssam@sfu.ca.

ABSTRACT
The RNA world hypothesis states that during an early stage of life, RNA molecules functioned as genome and as the only genome-encoded catalyst. This hypothesis is supported by several lines of evidence, one of which is the in vitro selection of catalytic RNAs (ribozymes) in the laboratory for a wide range of reactions that might have been used by RNA world organisms. This review focuses on three types of ribozymes that could have been involved in the synthesis of RNA, the core activity in the self-replication of RNA world organisms. These ribozyme classes catalyze nucleoside synthesis, triphosphorylation, and the polymerization of nucleoside triphosphates. The strengths and weaknesses regarding each ribozyme's possible function in a self-replicating RNA network are described, together with the obstacles that need to be overcome before an RNA world organism can be generated in the laboratory.

No MeSH data available.


Related in: MedlinePlus