Measuring the effects of amber initiator tRNA expression on a genomically recoded organism
Department of Molecular Sciences, Macquarie University, Sydney, NSW.
Bacterial translation initiation occurs at conserved AUG start codons recognized by the initiator tRNA anticodon. Since this codon-anticodon complementarity is sufficient for translation initiation, a mutant initiator tRNA (amber initiator) with the anticodon that recognizes an amber (UAG) stop codon has been shown previously to initiate translation at UAG start codons. Despite excellent prior work on characterizing the amber initiator, questions remain around the dynamic and population level behavior of cells carrying the amber initiator. To understand how the amber initiator impacts a cell with a reduced genetic code, I created a new modular set of two plasmids to be expressed in a genomically recoded organism E. coli strain C321.ΔA.exp. The plasmid set consists of an inducible system to express amber initiator and series of fluorescent reporter plasmids. This plasmid set has enabled me to show for the first time dynamic population-level characteristics of amber tRNA translation initiation. Fluorescence measurements confirmed that amber initiator starts translation from UAG start codon with 200-fold increase on inducing amber initiator compared to the repressed condition with 30-fold increase in reporter expression from an AUG start codon in similar conditions. Time-course measurements indicate different initiation effects as amber initiator matures. Surprisingly, proteomic analysis of cells expressing amber initiator showed no evidence of translation initiation from genomic UAG codons. These data suggest that the reduced genetic code of E. coli strain C321.ΔA.exp and conserved elements of amber initiator effectively prevent arbitrary initiation events from a UAG codon. However, proteomic analysis did reveal dramatic up-regulation of RNA-binding proteins. Lastly, I show that expressing amber initiator led to a reduced growth rate and reduced maximal cell density, revealing a link between phenotypic and proteomic effects.