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 this tRNA’s orthogonality. Here, we combine for the first time the amber initiator
in C321.ΔA.exp and measure its cellular effects. We found that the
expression resulted in a nearly 200Yfold increase in fluorescent reporter expression with a unimodal population distribution and no apparent cellular fitness defects. Proteomic analysis revealed upregulated ribosomeYassociated, tRNA degradation, and amino acid biosynthetic proteins, with no evidence for offYtarget translation initiation. In contrast to previous work, we show that UAGYinitiated proteins carry NYterminal methionine exclusively. Together, our results identify beneficial features of using the amber initiator
to control gene expression while also revealing fundamental challenges to using engineered initiator tRNAs as the basis for orthogonal translation initiation systems.
ABBREVIATIONS
- amber initiator tRNA with CUA anticodon
- SWATHYMS
- Sequential Window Acquisition of all Theoretical fragment ion Mass Spectra
- IF1
- Initiation Factor 1
- IF2
- Initiation Factor 2
- IF3
- Initiation Factor 3
- sfGFP(AUG)
- superfolder green fluorescence protein gene with wildYtype AUG start codon
- sfGFP(UAG)
- superfolder green fluorescence protein gene with amber stop codon UAG as start codon
- NanoLuc(AUG)
- the NanoLuc gene with wildYtype AUG start codon
- NanoLuc(UAG)
- the NanoLuc gene with amber stop codon UAG as start codon