Measuring Amber Initiator tRNA Orthogonality in a Genomically Recoded Organism.

@article{Vincent2019MeasuringAI,
  title={Measuring Amber Initiator tRNA Orthogonality in a Genomically Recoded Organism.},
  author={Russel M Vincent and Bradley W. Wright and Paul R. Jaschke},
  journal={ACS synthetic biology},
  year={2019},
  volume={8 4},
  pages={
          675-685
        }
}
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… 

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References

SHOWING 1-10 OF 55 REFERENCES
Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation
TLDR
The first attempts to reassign the sense methionine (Met) codon AUG to two different ncAAs in bacterial protein translation are described, suggesting that in vivo AUG codon reassignment is possible.
Genomically Recoded Organisms Expand Biological Functions
TLDR
The construction and characterization of a genomically recoded organism (GRO) is described, which exhibited improved properties for incorporation of nonstandard amino acids that expand the chemical diversity of proteins in vivo and exhibited increased resistance to T7 bacteriophage, demonstrating that new genetic codes could enable increased viral resistance.
Development of Assay Systems for Amber Codon Decoding at the Steps of Initiation and Elongation in Mycobacteria
TLDR
It is shown that formylation of the initiator tRNA (i-tRNA) is crucial even for slow-growing bacteria and that i-t RNA mutants with a CUA anticodon are aminoacylated by nondiscriminating GluRS.
The Fate of the Initiator tRNAs Is Sensitive to the Critical Balance between Interacting Proteins*
TLDR
It is shown that the relative levels of PTH and IF2 influence the path adopted by the initiator tRNAs in protein synthesis, and this findings provide an important clue to understand the dual function of the single tRNAMet in initiation and elongation, in the mitochondria of various organisms.
From elongator tRNA to initiator tRNA.
We show that the two most important properties needed for a tRNA to function in initiation in Escherichia coli are its ability to be formylated and its ability to bind to the ribosomal P site. This
Initiation of protein synthesis from a termination codon.
TLDR
Immunoblot analyses and assays for CAT enzyme activity in extracts from transformed cells indicate that initiation from UAG is efficient, 60-70% of that obtained from AUG, and initiation of protein synthesis with UAG and tRNA(fMetCUA) most likely occurs with glutamine and not methionine.
Role of methionine and formylation of initiator tRNA in initiation of protein synthesis in Escherichia coli
TLDR
The gene for E. coli methionyl-tRNA synthetase (MetRS) rescues, partially, the initiation defect of the mutant tRNA and a subset of mutant tRNAs which are defective in formylation and therefore inactive in initiation when they are aminoacylated with glutamine become partially active when MetRS is overproduced.
Avoidance of truncated proteins from unintended ribosome binding sites within heterologous protein coding sequences.
TLDR
It is found that balancing promoter strengths and upstream RBS strengths to intermediate levels can achieve the target protein concentration while avoiding both excessive noise and truncated protein.
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