Autonomous Biogenesis of Early and Late Assembly Intermediates of a Large Ribosomal Subunit in a Minimal System

Cell-free autonomous biogenesis of ribosomes is important for the bottom-up assembly of an artificial cell and for engineering of functional ribosomes with novel activities. The challenge lies in establishing conditions for a single-step multicomponent synthesis and assembly process in a minimal transcription–translation reaction. We previously reported autonomous synthesis and assembly of a bacterial small ribosomal subunit, but the assembly pathway of the large subunit is more complex with dozens of proteins that bind, fold, and chemically modify its ∼3000-base-long rRNA (rRNA). Here, we cosynthesized all the large subunit components including 33 ribosomal proteins (r-proteins) and 2 rRNAs from DNA brushes and followed the dynamics of assembly coupled to synthesis in real time using total internal reflection fluorescence microscopy. A systematic screen of the binding of each of the ribosomal proteins to the 23S rRNA revealed an assembly pattern indicative of early intermediates. The addition of genes coding for six rRNA assembly factors facilitated binding of late assembly ribosomal proteins, supporting formation of late-stage intermediates even in the absence of rRNA modifications. We established a scenario for the large subunit biogenesis via macromolecular colocalization that may aid in the identification and characterization of additional genes required for achieving complete functional ribosome self-regeneration in a minimal system.