Abstract
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 chip. Surface confinement provided favorable conditions for autonomous stepwise 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 toward creation of a self-replicating artificial cell and a general strategy for the mechanistic investigation of diverse multicomponent macromolecular machines.