Defects in the assembly of ribosomes selected for β-amino acid incorporation [article]

Fred R Ward, Zoe L Watson, Omer Ad, Alanna Schepartz, Jamie H. D. Cate
2019 bioRxiv   pre-print
Ribosome engineering has emerged as a promising field in synthetic biology, particularly concerning the production of new sequence-defined polymers. Mutant ribosomes have been developed that improve the incorporation of several non-standard monomers including D-amino acids, dipeptides, and β-amino acids into polypeptide chains. However, there remains little mechanistic understanding of how these ribosomes catalyze incorporation of these new substrates. Here we probed the properties of a mutant
more » ... erties of a mutant ribosome-P7A7-evolved for better in vivo β-amino acid incorporation through in vitro; biochemistry and cryo-electron microscopy. Although P7A7 is a functional ribosome in vivo, it is inactive in vitro,and assembles poorly into 70S complexes. Structural characterization revealed large regions of disorder in the peptidyl transferase center and nearby features, suggesting a defect in assembly. Comparison of RNA helix and ribosomal protein occupancy with other assembly intermediates revealed that P7A7 is stalled at a late stage in ribosome assembly, explaining its weak activity. These results highlight the importance of ensuring efficient ribosome assembly during ribosome engineering towards new catalytic abilities.
doi:10.1101/733584 fatcat:ocvxwqa45zbt3f5zlojz6w2v5m