Measuring amber initiator tRNA orthogonality in a genomically recoded organism

Research output: Contribution to journalLetterResearchpeer-review

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" tRNACUAfMet 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 tRNACUAfMet in C321.ΔA. exp and measure its cellular effects. We found that the tRNACUAfMet expression resulted in a nearly 200-fold increase in fluorescent reporter expression with a unimodal population distribution and no apparent cellular fitness defects. Proteomic analysis revealed upregulated ribosome-associated, tRNA degradation, and amino acid biosynthetic proteins, with no evidence for off-target translation initiation. In contrast to previous work, we show that UAG-initiated proteins carry N-terminal methionine, but have no evidence for glutamine. Together, our results identify beneficial features of using the amber initiator tRNACUAfMet to control gene expression while also revealing fundamental challenges to using engineered initiator tRNAs as the basis for orthogonal translation initiation systems.
LanguageEnglish
Pages675-685
Number of pages11
JournalACS Synthetic Biology
Volume8
Issue number4
Early online date11 Mar 2019
DOIs
Publication statusPublished - 2019

Fingerprint

RNA, Transfer, Met
Amber
Terminator Codon
Proteins
Amino Acid-Specific Transfer RNA
Transfer RNA
Housekeeping
Protein Biosynthesis
Population distribution
Glutamine
Ribosomes
Recombinant Proteins
Methionine
Proteomics
Metabolism
Demography
Gene expression
Escherichia coli
Gene Expression
Amino acids

Keywords

  • amber suppression
  • start codon
  • tRNA
  • orthogonality
  • translation

Cite this

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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{"} tRNACUAfMet 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 tRNACUAfMet in C321.ΔA. exp and measure its cellular effects. We found that the tRNACUAfMet expression resulted in a nearly 200-fold increase in fluorescent reporter expression with a unimodal population distribution and no apparent cellular fitness defects. Proteomic analysis revealed upregulated ribosome-associated, tRNA degradation, and amino acid biosynthetic proteins, with no evidence for off-target translation initiation. In contrast to previous work, we show that UAG-initiated proteins carry N-terminal methionine, but have no evidence for glutamine. Together, our results identify beneficial features of using the amber initiator tRNACUAfMet to control gene expression while also revealing fundamental challenges to using engineered initiator tRNAs as the basis for orthogonal translation initiation systems.",
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Measuring amber initiator tRNA orthogonality in a genomically recoded organism. / Vincent, Russel M.; Wright, Bradley W.; Jaschke, Paul R.

In: ACS Synthetic Biology, Vol. 8, No. 4, 2019, p. 675-685.

Research output: Contribution to journalLetterResearchpeer-review

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