Autonomous biogenesis of a ribosomal subunit on a chip

Reconstitution of self-replication of ribosomes is rudimentary for the establishment of a minimal artificial model system of a living cell. To make a new ribosome, an existing ribosome needs to synthesize about 60 different ribosomal proteins and assembly chaperons, coupled to transcription of 3 ribosomal RNAs, all coalescing to the unique structure of the ribosome in a sophisticated hierarchal assembly pathway. In the absence of crowding, confinement and cellular organization, the assembly of cell-free synthesized ribosomal components is challenging.

We established an experimental platform to demonstrate autonomous synthesis and assembly of ribosomes in a minimal cell-free reaction on a chip. Using surface-immobilized genes and real-time fluorescence measurements, we recreated the hierarchal assembly and timeline of the E. coli small ribosomal subunit and demonstrated its specific binding to surface-immobilized large ribosomal subunit1. Preliminary results suggest that the cell-free biogenesis of the large subunit is feasible. Our methodology utilizes synthetic genes, providing a biosafe procedure to investigate assembly and disruption of ribosomes and other multi-component bio-machines from diverse organisms and pathogens.

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 6, (2020).