National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM R01-114454
米国
National Science Foundation (NSF, United States)
1106400
米国
引用
ジャーナル: Biochemistry / 年: 2019 タイトル: Defects in the Assembly of Ribosomes Selected for β-Amino Acid Incorporation. 著者: Fred R Ward / Zoe L Watson / Omer Ad / Alanna Schepartz / Jamie H D Cate / 要旨: 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 ...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 β-amino acid incorporation through biochemistry and cryo-electron microscopy. Although P7A7 is a functional ribosome , it is inactive , 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.