Figure 3.

Transposase- lacZ translational and transcriptional fusion reporter assays in wt , dam ⁻ and hfq ⁻ strains. (A) Schematic of the IS50-lacZ transcriptional fusion (TCF; upper) and translational fusion (TLF; lower) reporters. The TCF reporter encodes the first 80 bp of IS50-Right (white rectangle) fused to lacZ (light blue rectangle). This fusion encodes only the first 15 nucleotides of the transposase (T1) transcript, which is expressed from the native promoter; the -35/-10 elements are shown in black. The inhibitor transcript is not expressed as the promoter for the inhibitor is missing its -10 region. The TLF encodes the first 128 bp of IS50-Right. This includes up to the 12^th codon of T1, which is fused in-frame to the 10^th codon of lacZ (purple rectangle). T1 and T2 and their respective promoter elements (-35/-10 sequences) are color-coded. Note that the start codon for the inhibitor protein has been mutated so that only transposase expression will give rise to β-galactosidase activity. Also note that the transposase promoter in both the TCF and the TLF is sensitive to Dam methylation. (B) β-galactosidase activity (given in Miller Units) for isogenic strains (wt, dam ⁻ or hfq ⁻) harboring either the TCF or TLF in single-copy in the chromosome of E. coli. For each fusion, the activity was normalized to that of the wt strain. The data sets shown for the TCF and TLF were compiled from two and three independent experiments, respectively, with each experiment including at least three replicates. Mean and standard error values are shown.