Measuring Amber Initiator tRNA Orthogonality in a Genomically Recoded Organism.

  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},
  volume={8 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" tRNACUAfMet that activates translation at UAG codons is available, but little is known about this… 

Figures and Tables from this paper

Orthogonal translation initiation using the non-canonical initiator tRNA(AAC) alters protein sequence and stability in vivo
The 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.
Initiation of Protein Synthesis with Non-canonical Amino Acids In Vivo.
It is demonstrated that itRNATy2 can initiate translation in vivo with aromatic non-canonical amino acids (ncAAs) bearing diverse sidechains and provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.
Suppression of Formylation Provides an Alternative Approach to Vacant Codon Creation in Bacterial In Vitro Translation
This work targeted the initiation machinery of E. coli, showing that restriction of the formyl donor or inhibition of theformyl transferase during in vitro translation is sufficient to prevent formylation of the acylated initiating tRNA and thereby create a vacant initiation codon that can be reprogrammed by exogenously charged tRNA.
Hijacking Translation Initiation for Synthetic Biology
Various methods which have been used to initiate protein synthesis with diverse molecules both in“vitro and in vivo” are explored.
A Codon Constrained Method for Both Eliminating and Creating Intragenic Bacterial Promoters
COdon Restrained Promoter SilEncing (CORPSE), a system for removing intragenic promoters, is presented and an inverted CORPSE system is presented, which can create highly active promoters within a gene sequence while not perturbing the function of the modified gene.
A high-resolution map of bacteriophage øX174 transcription
Next-generation sequencing is used to measure the RNA produced fromacteriophage øX174 while infecting its host E. coli C. coli and provides evidence for the first antisense transcription observed in the Microviridae family.


Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation
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
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
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*
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.
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
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.
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.