Fig. 1.

Coupled di-cistronic reporter system and initiator tRNAs. A. Schematic of the M13 geneV–geneVII (gV–gVII) intercistronic region fused in frame to chloramphenicol acetyltransferase (CAT) and firefly luciferase (fLuc) reporter genes, respectively, separated by a single C nucleotide. The stop and start codons for the CAT and fLuc reporters, respectively, are underlined. When required, the nucleotides in italics were changed to GA to create a Shine–Dalgarno sequence (GAGG) upstream of the fLuc gene. B. Structure of wild-type initiator tRNA₂^fMet and anticodon mutants that decode UAG (U35A36 mutant) and GUC (G34C36 mutant) start codons with the resulting changes in aminoacylation. C. Schematic of wild-type and mutant, di-cistronic and mono-cistronic reporters. Capital letters refer to the specific reporter gene (C, CAT; L, Luciferase), while subscript acronyms refer to specific alterations in the reporters as indicated.