Repurposing Ribosomes for Synthetic Biology: Ribo-T v2.0

Our recent advancement engineering a ribosome with tethered subunits, Ribo-T, represents a paradigm shift in the ability to engineer the key catalyst in gene expression, the ribosome (Nature, 524, 119-124, 2015; ACS Synth. Biol., 4, 1173-1175, 2015). Across all kingdoms of life, the ribosome is composed of two separate, completely dissociable subunits. Extensive ribosome engineering in vivo was limited by the requirement for ribosomes to maintain life. By mutating the mRNA ribosome binding site and the corresponding site on the small subunit, specialized orthogonal small subunits are created that operate in parallel to the wild-type pool; since the cell is not dependent on these orthogonal small subunits for life, their function can be altered in extraordinary ways. However, the large subunit, which contains the key engineering targets of the peptidyl transferase center and the nascent polypeptide exit tunnel, was not orthogonal due to promiscuous exchange between the wild-type and orthogonal small subunit pools. We were the first to solve this problem by tethering the large subunit to the small subunit via two short RNA linkers to create Ribo-T. While this first version of Ribo-T represents a step-change in our ability to engineer the ribosome and gives great promise for advancing basic science and synthetic biology, several limitations need to be overcome to develop it into a versatile tool with broader impact. Here, we use knowledge-driven and evolution-based approaches to improve Ribo-T properties, including orthogonal function. The new Ribo-T version 2.0 will be more versatile and will serve as a better tool for many biotechnological, engineering and basic science applications.