Reconstitution of ribosome self-replication outside a living cell

Ribosome biogenesis, a recursive process of pre-existing ribosomes self-replicating nascent ones, is pivotal in the self-replication of life. In Escherichia coli, three ribosomal RNAs (rRNAs) are transcribed, and 54 ribosomal proteins (r-proteins) are synthesized by pre-existing ribosomes as structural components1, 2. They are cotranscriptionally assembled in a cooperative hierarchy under the support of [~]100 accessory factors1-3. The reconstitution of ribosome biogenesis outside a living cell is an essential goal to understand the self-replication of life. However, this goal could not have been achieved so far due to its complexity. Here, we report the successful in vitro reconstitution of the entire ribosome biogenesis process. We hypothesized that mimicking in vivo ribosome biogenesis1-6 could result in in vitro ribosome biogenesis. Specifically, we found that coactivating the transcription of an rRNA operon, as well as the transcription and translation of 54 r-protein genes encoding r-proteins, and the coordinated ribosomal assembly in a cytoplasm-mimicking reaction solution, resulted in highly efficient in vitro reconstitution of ribosome biogenesis. Our achievement represents a critical step toward revealing fundamental principles underlying the self-replication of life and creating self-replicating artificial cells7. We also succeeded in engineering rRNA and r-proteins by only adding mutant ribosomal genes in the reaction, enabling high-throughput and unconstrained creation of artificial ribosomes with altered or enhanced functionality8-12.