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Measuring amber initiator tRNA orthogonality in a genomically recoded organism

View ORCID ProfileRussel M. Vincent, View ORCID ProfileBradley W. Wright, View ORCID ProfilePaul R. Jaschke
doi: https://doi.org/10.1101/524249
Russel M. Vincent
†Department of Molecular Sciences, Macquarie University, Sydney 2109, New South Wales, Australia
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Bradley W. Wright
†Department of Molecular Sciences, Macquarie University, Sydney 2109, New South Wales, Australia
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Paul R. Jaschke
†Department 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

Using engineered initiator tRNA for precise control of protein translation within cells has great promise within future orthogonal translation systems to decouple housekeeping protein metabolism from that of engineered genetic systems. Previously, E. coli strain C321.ΔA.exp lacking all UAG stop codons was created, freeing this ‘amber’ stop codon for other purposes. An engineered ‘amber initiator’ Embedded Image that activates translation at UAG codons is available, but little is known about this tRNA’s orthogonality. Here, we combine for the first time the amber initiator Embedded Imagein C321.ΔA.exp and measure its cellular effects. We found that the Embedded Imageexpression resulted in a nearly 200Yfold increase in fluorescent reporter expression with a unimodal population distribution and no apparent cellular fitness defects. Proteomic analysis revealed upregulated ribosomeYassociated, tRNA degradation, and amino acid biosynthetic proteins, with no evidence for offYtarget translation initiation. In contrast to previous work, we show that UAGYinitiated proteins carry NYterminal methionine exclusively. Together, our results identify beneficial features of using the amber initiator Embedded Imageto control gene expression while also revealing fundamental challenges to using engineered initiator tRNAs as the basis for orthogonal translation initiation systems.

  • ABBREVIATIONS

    Embedded Image
    amber initiator tRNA with CUA anticodon
    SWATHYMS
    Sequential Window Acquisition of all Theoretical fragment ion Mass Spectra
    IF1
    Initiation Factor 1
    IF2
    Initiation Factor 2
    IF3
    Initiation Factor 3
    sfGFP(AUG)
    superfolder green fluorescence protein gene with wildYtype AUG start codon
    sfGFP(UAG)
    superfolder green fluorescence protein gene with amber stop codon UAG as start codon
    NanoLuc(AUG)
    the NanoLuc gene with wildYtype AUG start codon
    NanoLuc(UAG)
    the NanoLuc gene with amber stop codon UAG as start codon
  • Copyright 
    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-NC-ND 4.0 International license.
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    Posted January 18, 2019.
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    Measuring amber initiator tRNA orthogonality in a genomically recoded organism
    Russel M. Vincent, Bradley W. Wright, Paul R. Jaschke
    bioRxiv 524249; doi: https://doi.org/10.1101/524249
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    Measuring amber initiator tRNA orthogonality in a genomically recoded organism
    Russel M. Vincent, Bradley W. Wright, Paul R. Jaschke
    bioRxiv 524249; doi: https://doi.org/10.1101/524249

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