Title:
Engineering elongation factor Tu and tRNAs to better accommodate non-canonical amino acids during translation

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DeLey Cox, Vanessa E.
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Gaucher, Eric A.
Williams, Loren D.
Finn, M. G.
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Abstract
Non-canonical amino acid (ncAA) incorporation has led to significant advances in protein science and engineering. Traditionally, incorporation of ncAAs is achieved via amber codon suppression using an engineered orthogonal aminoacyl-tRNA synthetase:tRNA pair. However, as more complex protein products are targeted, researchers are identifying additional barriers limiting the scope of currently available ncAA systems. One barrier is elongation factor Tu (EF-Tu), a protein responsible for proof-reading aa-tRNAs that substantially restricts ncAA scope by limiting ncaa-tRNA delivery to the ribosome. Researchers have responded by engineering ncAA-compatible EF-Tus for key ncAAs. However, this approach fails to address the extent to which EF-Tu inhibits efficient ncAA incorporation. Herein, we describe our efforts to transform how EF-Tu is utilized to incorporate ncAAs. Leveraging computational methods, we present data on four EF-Tu libraries and two tRNA libraries used in both in vitro and in vivo translation. Ultimately, the in vivo assay led to the identification of EF-Tu variants which accept non-native substrates. By mass spectrometry, we demonstrate two variants have expanded substrate acceptance. Fitness tests show a polyspecific EF-Tu is an accepted addition to the translation machinery and improves host organism fitness relative to an ncAA-specific engineered EF-Tu. These results lend credence to our choice of computational method (in this case REAP) and also suggest that EF-Tu and SelB may have a shared, substrate-promiscuous ancestor. Overall, we believe this approach complements current research highlighting the advantages of improved OTSs and promotes a more comprehensive approach critical to achieving future goals.
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2018-07-26
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Dissertation
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