(78d) In Vitro Ribosome Construction for Ribosome Engineering

The ribosome is a complex macromolecular machine responsible for synthesis of proteins with diverse biological functions. The extraordinary synthetic capability of the ribosome has long made the isolation of engineered variants with altered functions an attractive prospect. For example, limited studies have shown that modifying ribosomes can enhance tolerance of unnatural amino acid substrates. Unfortunately, constraints imposed by living cells limit efforts to make engineered ribosomes in vivo. A simple, purely in vitro strategy for ribosome construction would offer a powerful solution for studying and exploiting modified ribosomes. To that end, we have developed an integrated synthesis, assembly, and translation technology (termed iSAT) for the in vitro construction of Escherichia coli ribosomes in crude S150 extracts. iSAT allows for the simultaneous synthesis of ribosomal RNA (rRNA), assembly of rRNA with purified ribosomal proteins, and translation of a reporter protein (e.g. luciferase) as a measure of ribosome activity in the same compartment. Here we report the improvement of iSAT activity by ~45-fold as compared to the previous state-of-the-art by tuning the transcriptional balance of rRNA and mRNA production. Specifically, modifications to the 3’ rRNA gene sequence to increase transcriptional efficiency, optimization of plasmid and polymerase concentrations, and use of native rRNA operon genes for stoichiometrically balanced rRNA synthesis and native rRNA processing resulted in increases in iSAT activity. With enhanced iSAT activity, we are now developing and will also report on a novel in vitro ribosome evolution strategy for ribosome engineering. Specifically, we are using iSAT to generate a library of ribosomes with rRNA mutations and selecting for functionality using ribosome display. Looking forward, we believe our platform will enable in vitro evolution to select for ribosomes that have enhanced functions or altered chemical properties.