Autonomous biogenesis of a ribosomal subunit on a chip

Ribosomes are the universal decoders of the genetic code and unique biological machines that synthesize their own parts and self-assemble in a sophisticated step-wise process. Ribosome assembly has been studied for decades to elucidate the composition of intermediates, assembly order, thermodynamics and kinetics of assembly, yet to date there is no in vitro reconstituted system of ribosomes synthesizing ribosomes.

We established an experimental scheme for autonomous self-synthesis of Escherichia coli‘s small ribosomal subunit in a minimal cell-free reaction on a chip, allowing us to dynamically recreate its assembly pathway. Using surface-immobilized genes^1-3, our real-time fluorescence measurements revealed hierarchal assembly, cooperative interactions, unstable intermediates, and specific binding to large ribosomal subunits. Using only synthetic genes, we applied our cell-free approach to the elucidation ofthe unknown assembly pathway of the human pathogen Staphylococcus aureus’s small ribosomal subunit in a completely biosafe procedure, revealing a kinetic role of protein assembly-chaperons. This 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 bio-machines⁴.

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