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bioRxiv · 10.1101/733584

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

Ward, F. R.·Watson, Z. L.·Ad, O.·Schepartz, A.·Cate, J. H. D.
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Abstract

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 {beta}-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 ribosome-P7A7-evolved for better in vivo {beta}-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 peptidyltransferase 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.

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Ward, F. R., Watson, Z. L., Ad, O., Schepartz, A., Cate, J. H. D.. 2019-08-13. Defects in the assembly of ribosomes selected for β-amino acid incorporation. https://doi.org/10.1101/733584

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