Coupled catabolism and anabolism in autocatalytic RNA sets

Abstract

The ability to process molecules available in the environment into useable building blocks characterizes catabolism in contemporary cells and was probably critical for the initiation of life. Here we show that a catabolic process in collectively autocatalytic sets of RNAs allows diversified substrates to be assimilated. We modify fragments of the Azoarcus group I intron and find that the system is able to restore the original native fragments by a multi-step reaction pathway. This allows in turn the formation of catalysts by an anabolic process, eventually leading to the accumulation of ribozymes. These results demonstrate that rudimentary self-reproducing RNA systems based on recombination possess an inherent capacity to assimilate an expanded repertoire of chemical resources and suggest that coupled catabolism and anabolism could have arisen at a very early stage in primordial living systems.

Figure 1.

Coupled catabolism and anabolism in self-reproducing systems. (A) Schematic showing how catabolism in a self-reproducing system can process unusable raw material into usable substrates. (B) Autocatalytic synthesis of covalent RNA catalyst (WXYZ) from inactive RNA substrates (WXY and Z) using an anabolic autocatalytic process in the Azoarcus ribozyme system.

Figure 2.

Synthesis of WXYZ catalyst with modified RNA substrates. (A) Different substrate combinations used to study the catabolic properties of the Azoarcus ribozyme system. (B) Kinetics of the synthesis of WXYZ using all four substrate combinations (c1 to c4). The reported yield is the substrate to product conversion in percentage. Error bars represent ±1 standard deviation (triplicates).

Figure 3.

Multi-step reaction pathway to catabolize the starting material. (A) Diagrammatic representation of the catabolic steps involved in the synthesis of substrates WXY and Z from the modified RNAs (WXY-mod and Z-mod) to produce WXYZ. Dotted and continuous gray arrow show feed-back by non-covalent and covalent catalyst, respectively. (B) Kinetic analysis of the formation of WXY-mod with substrate combination c2. (C) Kinetic analysis of the formation of WXY from WXY-mod with substrate combination c3. For both graphs (B) and (C), the circles represent experimental data obtained by polyacrylamide gel electrophoresis and the lines represents the data obtained from kinetic modeling (Supplementary Text). The reported yield is the substrate to product conversion in percentage. Error bars represent ±1 standard deviation (triplicates).

Figure 4.

Proposed binding of Z and Z-mod and effect of mutations in Z-mod. (A) Diagrammatic representation of the binding of Z to WXY in the case of non-modified substrates (20,29). (B) Proposed binding of Z-mod to WXY. (C) Sequence of mutated variants (M1-6, substitutions in red) of Z-mod and the yield of WXY-mod after 1 h reaction between WXY and non-mutated or mutated variants of Z-mod obtained by polyacrylamide gel electrophoresis. The reported yield is the substrate to product conversion in percentage. Error bars represent ±1 standard deviation (triplicates).

Figure 5.

Formation of cooperative RNA network using modified substrates. Reactions were started by adding WXY fragments with different IGS-tag sequences (MN = AA, GU and UC, right panel) as used in previous studies (30,31). The reaction is either provided with native (Z, purple) or modified substrate (Z-mod, orange). Kinetic analysis was performed by analysing the formation of WXYZ at different time points using polyacrylamide gel electrophoresis. The reported yield is the substrate to product conversion in percentage. Error bars represent ±1 standard deviation (triplicates).