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Orthogonal translation initiation using the non-canonical initiator tRNA(AAC) alters protein sequence and stability in vivo

View ORCID ProfileAndras Hutvagner, View ORCID ProfileDominic Scopelliti, View ORCID ProfileFiona Whelan, View ORCID ProfilePaul R. Jaschke
doi: https://doi.org/10.1101/2021.05.25.445580
Andras Hutvagner
1Department of Molecular Sciences, Macquarie University, Sydney 2109, New South Wales, Australia
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Dominic Scopelliti
1Department of Molecular Sciences, Macquarie University, Sydney 2109, New South Wales, Australia
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Fiona Whelan
2Department of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, South Australia, Australia
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Paul R. Jaschke
1Department of Molecular Sciences, Macquarie University, Sydney 2109, New South Wales, Australia
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  • For correspondence: paul.jaschke@mq.edu.au
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Abstract

Biological engineers seek to have better control and a more complete understanding of the process of translation initiation within cells so that they may produce proteins more efficiently, as well as to create orthogonal translation systems. Previously, initiator tRNA variants have been created that initiate translation from non-AUG start codons, but their orthogonality has never been measured and the detailed characteristics of proteins produced from them have not been well defined. In this study we created an initiator tRNA mutant with anticodon altered to AAC to be complementary to GUU start codons. We deploy this i-tRNA(AAC) into E. coli cells and measure translation initiation efficiency against all possible start codons. Using parallel reaction monitoring targeted mass spectrometry we identify the N-terminal amino acids of i-tRNA(AAC)-initiated reporter proteins and show these proteins have altered stability within cells. We also use structural modeling of the peptide deformylase enzyme interaction with position 1 valine peptides to interrogate a potential mechanism for accumulation of formylated-valine proteins observed by mass spectrometry. Our results demonstrate that mutant initiator tRNAs have potential to initiate translation more orthogonally than the native initiator tRNA but their interactions with cellular formyltransferases and peptide deformylases can be inefficient because of the amino acid they are charged with. Additionally, engineered initiator tRNAs may enable tuning of in vivo protein stability through initiation with non-methionine amino acids that alter their interaction with cellular proteases.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Posted May 25, 2021.
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Orthogonal translation initiation using the non-canonical initiator tRNA(AAC) alters protein sequence and stability in vivo
Andras Hutvagner, Dominic Scopelliti, Fiona Whelan, Paul R. Jaschke
bioRxiv 2021.05.25.445580; doi: https://doi.org/10.1101/2021.05.25.445580
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Orthogonal translation initiation using the non-canonical initiator tRNA(AAC) alters protein sequence and stability in vivo
Andras Hutvagner, Dominic Scopelliti, Fiona Whelan, Paul R. Jaschke
bioRxiv 2021.05.25.445580; doi: https://doi.org/10.1101/2021.05.25.445580

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