FIG. 2.
AlgZ binding is localized to a region spanning from −185 to −66 bp upstream of algZ. (A) The sequence of algZ (−278 to +109 bp) was divided into four overlapping fragments (fragments A to D). Fragment A encompassed sequences from −278 to −173 bp. Fragment B encompassed sequences from −185 to −66 bp. Fragment C encompassed sequences from −77 to +20 bp. Fragment D encompassed sequences from +10 to +109 bp. (B) DNA fragments were end labeled and incubated either alone (lanes 1, 4, 7, 10, and 13), with 0.5 μg of an extract derived from E. coli BL21(DE3)/pET29a (expression vector, lanes 2, 5, 8, 11, and 14), or with 0.5 μg of an extract derived from BL21(DE3)/pPJ145 (AlgZ, lanes 3, 6, 9, 12, and 15) in an EMSA. The amount of AlgZ used in this experiment was determined empirically and verified by phosphorimaging densitometry to be the amount of protein required to shift 89% of the free DNA containing all four fragments (A to D) (lane 3). Arrows indicate AlgZ-DNA complexes.