Autonomous Cell-Free Synthesis and Assembly of a Ribosomal Subunit

Ribosome biogenesis is an efficient and complex assembly process that has not been reconstructed outside a living cell so far, yet is the most critical step for establishing a self-replicating artificial cell. We recreated the biogenesis of Escherichia coli’s small ribosomal subunit by synthesizing and capturing all its ribosomal proteins and RNA on a chip¹. Surface confinement provided favorable conditions for autonomous step-wise assembly of new subunits, spatially segregated from original intact ribosomes. Our real-time fluorescence measurements revealed hierarchal assembly, cooperative interactions, unstable intermediates, and specific binding to large ribosomal subunits. Using only synthetic genes, our methodology is a crucial step towards creation of a self-replicating artificial cell and a general strategy for the mechanistic investigation of diverse multi-component macromolecular machines.

(1) Levy, M.; Falkovich, R.; Daube, S. S.; Bar-Ziv, R. H. Autonomous Synthesis and Assembly of a Ribosomal Subunit on a Chip. Sci. Adv. 2020, 6 (16), eaaz6020. https://doi.org/10.1126/sciadv.aaz6020.