Emergence of linkage between cooperative RNA replicators encoding replication and metabolic enzymes thorough experimental evolution

Abstract

The integration of individually replicating genes into a primitive chromosome is a key evolutionary transition in the development of life, allowing the simultaneous inheritance of genes. However, how this transition occurred is unclear because of the extended size of primitive chromosomes, which replicate slower than unlinked genes. Theoretical studies have suggested that a primitive chromosome can evolve in the presence of cell-like compartments, as the physical linkage prevents the stochastic loss of essential genes upon division, but experimental support for this is lacking. Here, we demonstrate the evolution of a chromosome-like RNA from two cooperative RNA replicators encoding replication and metabolic enzymes. Through their long-term replication in cell-like compartments, linked RNAs emerged with the two cooperative RNAs connected end-to-end. The linked RNAs had different mutation patterns than the two unlinked RNAs, suggesting that they were maintained as partially distinct lineages in the population. Our results provide experimental evidence supporting the plausibility of the evolution of a primitive chromosome from unlinked gene fragments, an important step in the emergence of complex biological systems.

Cell-free expression of RuBisCO for ATP production in the synthetic cells

“…The reaction mixture contained 50 mM HEPES–KOH (pH 7.8), 10 mM NaHCO 3 , 20 mM MgCl 2 , 0.2 mM NADH, 5 mM ATP, 5 mM phosphocreatine, 5 U creatine phosphokinase, 5 U glyceraldehyde-3-phosphate dehydrogenase (Sigma-Aldrich G2267), 5 U 3-phosphoglycerate kinase (Sigma-Aldrich P7634). The creatine phosphokinase was purified previously as a component of the customized PURE system ( 30 ). The absorbance of the reaction solution was then measured using a spectrophotometer (UV-2550, SHIMADZU) under two conditions: 0 min and 30 min incubated at 30°C.…”

“…The customized PURE system used here consists of highly purified protein components to avoid kinase contamination. Each component was purified through additional column purification in a stringent buffer as described previously ( 30 ). After incubation, 1 µl aliquots of the reaction mixture were diluted 50-fold with a luciferase-based ATP assay kit (Fujifilm-Wako, 340–09791), inverted four times, and immediately measured for oxyluciferin emission using a Luminometer (GloMax, Promega).…”