A System for Unprecedented Ribosome Engineering in Living E. coli

The Escherichia coli ribosome is a 2.4 MDa molecular machine that consists of a large subunit and a small subunit, and is the key catalyst in gene expression. It is responsible for synthesizing proteins from natural amino acids in a sequence-defined fashion with impressive speed and accuracy. Expanding the repertoire of ribosome substrates and functions would be greatly beneficial for the advancement of systems and synthetic biology. However, as with any biological system, engineering objectives are often completely opposed to the growth and reproduction objectives of the organism. This problem can be solved through the use of a specialized ribosome that translates only a specific type of engineered messenger RNAs (mRNAs) and avoids translation of native cellular mRNAs. So far, efforts to construct such orthogonal ribosomes have focused on modifying the small subunit alone because orthogonality is endowed by modifying the Shine-Dalgarno sequence of an mRNA and the corresponding complementary sequence in the 16S ribosomal RNA (rRNA) of the small subunit. Unfortunately, free exchange between the subunits meant the large subunit, which is responsible for peptide bond formation and protein excretion, could not be extensively engineered. Here we address this challenge. Specifically, we show that an engineered ribosome with tethered subunits (termed Ribo-T), which contains a single hybrid rRNA composed of small and large subunit rRNA sequences, is capable of protein synthesis in vitro and in vivo. Considering that the ribosome is one of nature's most evolved, fine-tuned and conserved structures, it is surprising that we were able to engineer a ribosome with inseparable subunits. One of the exciting implications of Ribo-T is the possibility of introducing mutations in large ribosomal subunits that would be deleterious if introduced in an untethered ribosome (e.g., dominant lethal). We show the ability to evolve Ribo-T by selecting otherwise dominantly lethal rRNA mutations in the large ribosomal subunit that facilitate translation of challenging protein sequences. We anticipate that Ribo-T will advance fundamental understanding of the ribosome, enable dual translation systems in cells, and catalyze a new paradigm for synthesis and evolution of abiological polymers.