UC Berkeley

The ribosome catalyzes the synthesis of polypeptides with high efficiency and sequence specificity. Repurposing the ribosome as a platform for manufacturing other sequence defined polymers could access a wide variety of previously unattainable molecules and bulk materials. In this work, we aim to understand both engineered and wild type ribosomes through structural and biochemical analysis. We reveal limitations of previous engineering efforts on the ribosome, highlighting the importance of careful mutation and selection techniques. The ribosome we study is poorly assembled and nonfunctional in vitro despite improved polymerization of β-amino acids in vivo. We identify key regions of the ribosome that are disrupted by mutations and offer suggestions for more targeted engineering that will preserve efficient ribosome assembly. We also characterize the structure of the wild type ribosome bound to an unnatural, non-α-amino acid monomer for the first time. This monomer is correctly accommodated into the P site of the ribosome, explaining previously observed activity as an initiator substrate. Lastly, we assess a new, short peptide luciferase-complementing reporter in defined in vitro translations as a better readout of mutant ribosome activity. Using this assay, we show activity of purified active site mutant ribosomes in vitro for the first time, reconciling often observed differences from in vivo systems. These projects underscore the importance of detailed characterization of both the input to and results of ambitious bioengineering efforts.