Extended Data Fig. 1: Features and activity of the triplet polymerase ribozyme 5TU+1. | Nature Chemistry

Extended Data Fig. 1: Features and activity of the triplet polymerase ribozyme 5TU+1.

From: Trinucleotide substrates under pH–freeze–thaw cycles enable open-ended exponential RNA replication by a polymerase ribozyme

Extended Data Fig. 1

(a) A secondary-structure-level representation of the 5TU+1 (5TU/t1) triplet polymerase ribozyme (TPR) heterodimer based upon its cryo-EM structural model21. The TPR comprises a catalytic 5TU subunit (orange) and an inactive type 1 (t1) subunit (blue), depicted next to a template-substrate complex. Triplet substrate (green) binding to template (light grey) juxtaposes the 3’ hydroxyl of the upstream RNA primer (brown) and the 5’ triphosphate of the triplet (purple). The 5TU subunit catalyses nucleophilic attack of the 3’ hydroxyl on the triphosphate α-phosphate of a correctly-paired triplet to form a new phosphodiester linkage, repeating this process iteratively. Type 1 forms a heterodimer with 5TU, improving 5TU’s interaction with ligation junctions. The γ fragment of 5TU is highlighted in red, and type 1 regions complementary to products in Extended Data Fig. 8 are highlighted in lilac. (b) Primer extension by the TPR. Full-length product is generated when all junctions are ligated, and incomplete ligation yields a ladder of intermediate extension products. Here, multiple-turnover catalysis allows the TPR to synthesise complementary strand RNA upon excess primer/template molecules. 40 nM of single-stranded template A+ and primer FITCA were mixed with the indicated concentrations of TPR in 1 mM MgCl2, 1 mM CHES·KOH pH 9.0, 4 mM KCl, 0.001% Tween-20, and 0.1 µM of each triplet substrate. To initiate extension, the mixture was frozen in dry ice then incubated at −7 °C for 24 h. Formation of a supercooled liquid brine eutectic phase concentrated all solutes by an estimated 250-fold. Primer extension products were resolved by denaturing PAGE, and gel densitometry allowed quantification of extension and calculation of the number of complementary strands synthesised per ribozyme molecule. Considering that each strand is itself the product of eight iterative ligation reactions, at 0.4 nM TPR each TPR molecule must carry out a ligation every few minutes in ice.

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