Ribosome subunit stapling for orthogonal translation in E. coli

Abstract

The creation of orthogonal large and small ribosomal subunits, which interact with each other but not with endogenous ribosomal subunits, would extend our capacity to create new functions in the ribosome by making the large subunit evolvable. To this end, we rationally designed a ribosomal RNA that covalently links the ribosome subunits via an RNA staple. The stapled ribosome is directed to an orthogonal mRNA, allowing the introduction of mutations into the large subunit that reduce orthogonal translation, but have minimal effects on cell growth. Our approach provides a promising route towards orthogonal subunit association, which may enable the evolution of key functional centers in the large subunit, including the peptidyl-transferase center, for unnatural polymer synthesis in cells. Stapling the ribosome together: An orthogonal ribosome was designed in which the large and small subunits are connected through a covalent RNA staple, and directed to an orthogonal mRNA. This ribosome is active in protein synthesis in cells and enables otherwise lethal mutations to be introduced into the large subunit. This paves the way for the ribosome-catalyzed synthesis of unnatural polymers by cellular orthogonal translation.