Measuring Amber Initiator tRNA Orthogonality in a Genomically Recoded Organism

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.