FIG 2.

The distance of the ACA site from the AUG initiation codon plays a role in the expression of the stress-induced GFP reporter. (A) GFP reporter system for studying the effect of the location of ACA sites upstream from the AUG initiation codon on translation under stress. We used a gfp reporter gene that carries no ACA sites in its coding sequence and no ACA sites upstream (up to 135 nucleotides) from the ATG initiation codon (13). We used this reporter system as a basis for generating five new constructs. In each new construct, we inserted an ACA site upstream from the ATG initiation codon at specific locations, nucleotides 20, 40, 80, 100, and 120. 5’-UTR, 5′ untranslated region. (B) Levels of GFP expression as a result of the location of the ACA site upstream from the AUG initiation codon. E. coli strain MG1655 was separately transformed with plasmid pUH-C carrying five different gfp reporter genes with an ACA site upstream from the initiation codon, ATG. Here we show a quantitative comparison of levels of GFP expression in samples induced by nalidixic acid (green bars) and in uninduced samples (blue bars). These data were calculated as percentages of fluorescence units. For each assay, 100% represents the results for the untreated sample.