(328d) Towards Construction of a Synthetic Self-Replicating Entity

A powerful approach to enhance our understanding of complex biomolecular systems is to build integrated biological processes from their fundamental components. The long-term vision of this project is to construct a useful synthetic entity that self-replicates from small molecule building blocks and reconstitutes the macromolecular catalysts synthesizing DNA, RNA and protein. This objective demands in vitro methods for production and self-assembly of active component parts. Here we describe recent efforts towards constructing highly active E. coli ribosomes from their constituent parts. Previous works have shown total reconstitution of functionally active 30S and 50S subunits from native E. coli rRNA and proteins. However, reconstituted ribosomes have only ever been shown competent for their ability to translate poly-uridine [Poly(U)] mRNA templates into polyphenylalanine. Moreover, reconstituting E. coli ribosomes under physiological conditions has remained elusive for decades. Here we show that reconstituted ribosomes can synthesize active full-length protein, luciferase and green fluorescent protein. Furthermore, we show that active E. coli ribosomes can be reconstituted in a one-step incubation procedure at 37°C under conditions that mimic the cytoplasm. Finally, as a milestone towards building a synthetic replicon, we provide evidence that rRNA synthesis, ribosome assembly, and protein synthesis can be carried out in one compartment. Our reconstitution methods will contribute towards the in vitro analysis of ribosomal mutations for understanding the molecular details of ribosome function. Equally important, our approach solves one of the critical barriers to constructing ribosomes in vitro and promises to enable cell-free synthetic biology as a platform for evolving ribosomes for the production of protein therapeutics and peptide drugs that are difficult to make in vivo because of their toxicity, complexity, or unusual cofactor requirements.