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.