Reprogramming the genetic code: defining a new start codon in Escherichia coli

Translation maps triplet RNA codons onto single amino acids within a protein. Bacterial translation initiation occurs at a conserved AUG start codon recognised by the initiator tRNA anticodon, with studies demonstrating that codon-anticodon complementarity is sufficient for initiation. Thus far, engineering initiation codons have been neglected aside from one identified mutant amber initiator tRNA (tRNA CUAfmet2) that can start protein synthesis from the UAG stop codon. In this study, I create a new inducible system to express tRNA CUAfmet2 and a new set of fluorescent reporter plasmids to enable population-level studies for the first time. Fluorescence measurements confirmed that tRNA CUAfmet2 initiates translation from UAG start codon with 200-fold increase on inducing tRNA CUAfmet2 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 tRNA CUAfmet2 matures. Proteomic analysis of cells expressing tRNA CUAfmet2 reveal a minimal effect on the host proteome with no evidence of peptides initiating from genomic UAG codons. I demonstrate that my tRNA CUAfmet2-expressing plasmid is transportable in all five E. coli strains with no adverse fitness defects. In this thesis, I define tRNA CUAfmet characteristics with future scope for refinement for this orthogonal translation initiation system -- abstract.