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N-terminal amino acid identification using targeted mass spectrometry

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’ that activates translation at UAG codons is available, but little is known about amino acid it assigns to the amber start codon. Here, we combine for the first time the amber initiator tRNA in C321.ΔA.exp and measure its cellular effects. Using, targeted proteomics, we found that the amber initiator tRNA initiates proteins exclusively with methionine.

ORGANISM(S): Escherichia Coli

SUBMITTER: Russel Vincent

PROVIDER: PXD012390 | panorama | Fri Mar 15 00:00:00 GMT 2019

REPOSITORIES: PanoramaPublic

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Measuring Amber Initiator tRNA Orthogonality in a Genomically Recoded Organism.

Vincent Russel M RM Wright Bradley W BW Jaschke Paul R PR

ACS synthetic biology 20190315 4

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" tRNA<sub>CUA</sub><sup>fMet</sup> that activates translation at UAG codons is available, but little ...[more]

PMID: 30856316

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