Open AccessDefects in the Assembly of Ribosomes Selected for β-Amino Acid Incorporation
Author(s) -
Fred R. Ward,
Zoe L Watson,
Omer Ad,
Alanna Schepartz,
Jamie H. D. Cate
Publication year - 2019
Publication title -
biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.43
H-Index - 253
eISSN - 1520-4995
pISSN - 0006-2960
DOI - 10.1021/acs.biochem.9b00746
Subject(s) - ribosome , amino acid , peptidyl transferase , protein biosynthesis , ribosomal rna , biochemistry , eukaryotic ribosome , translation (biology) , biology , ribosomal protein , chemistry , microbiology and biotechnology , rna , biophysics , messenger rna , gene
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 nonstandard 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 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 ribosome 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 toward new catalytic abilities.