FIG. 3.
Effect of spermidine on P-PrrA and U-PrrA binding affinity in vitro. (A) EMSA results showing binding of increasing concentrations of P-PrrA to an ∼0.35-pmol labeled double-stranded 191-bp fragment containing the RSP3361 PrrA site 2. The reaction conditions are described in Materials and Methods. The analysis was performed in the presence (+SP) and absence (−SP) of 2 mM spermidine. Both gels were run identically. PrrA concentrations (μM) are indicated above the lanes. (B) Quantitation of unbound DNA as a function of P-PrrA concentration. The calculated values of 4.6 × 10−8 M (+SP) and 1.9 × 10−7 M (−SP) correspond to the P-PrrA concentrations at which 50% of the labeled DNA is unbound, or 0.5 fractional saturation. Poly(dA) · poly(dT) was added as a nonspecific competitor at 80 ng/μl concentration. (C) Effects of increasing concentrations of U-PrrA on U-PrrA-DNA complex formation in the presence or absence of salmon sperm DNA, with 2 mM spermidine added to the reactions. The plus sign refers to addition of 2 μg of salmon sperm DNA (67 ng/μl). The concentrations of U-PrrA (μM) are indicated above the lanes. In all panels, lane 1 contains DNA only. The minus sign indicates that there was no addition of the specific compound.