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 all its ribosomal proteins and RNA from surface-immobilized genes in a minimal cell-free reaction. Surface confinement created favorable biophysical conditions for concomitant synthesis and interactions of parts and for the visualization of nascent subunits, which were captured on the surface spatially segregated from original 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.