Fig 4.

Effects of substitution mutations in the yjiY promoter on in vitro YehT binding and YehT-dependent promoter activity in vivo. (A) The wild-type core region P_{yjiY P} (bp −101 to −44) (Fig. 3C) of the yjiY promoter, comprising motifs M1, M2, and M3, and several mutated variants of the same region were analyzed by EMSA following incubation with the indicated concentrations of 6His-YehT. In the mutant fragments, motifs M1 to M3 were systematically removed by substituting purines in the sequence for pyrimidines and vice versa. The resulting fragments were designated P_{yjiY MM3} (M3 site replaced), P_{yjiY MM23} (M2 and M3 replaced), and P_{yjiY MM123} (all three M sites replaced). In addition, the 14-bp spacer between M1 and M2 was mutated in the same way, resulting in fragment P_{yjiY SC14}. All fragments were analyzed by EMSAs. The positions of free DNA (filled arrows) and YehT-DNA complexes (open arrows) are marked. (B) Effects of the various substitutions within the yjiY promoter on YehT-dependent transcription in vivo. E. coli MG1655-ΔlacZ/pBAD18-yehT was transformed with plasmid pRS415 encoding various yjiY promoter∷lacZ fusions (see the text for detailed descriptions). Bacteria were cultivated under aerobic growth conditions in LB medium at 37°C until the exponential-growth phase. Then yehT overexpression was induced by adding 0.2% (wt/vol) l-arabinose, and cells were harvested after 45 min. β-Galactosidase (LacZ) activity was then determined as a measure of yjiY promoter activity. Experiments were performed at least three times, and error bars indicate the standard deviations of the means.