Figure 3. Phosphorylation-dependent binding of CpxR to DNA upstream of Yersinia virulence genes.

Mobility shift assays were performed with purified CpxR_wt::His₆, and the non-phosphorylated mutant CpxR_D51A::His₆. Target DNAs were radiolabeled PCR fragments harboring the regulatory regions of cpxR/cpxP, ppiA, rovA, inv, ail (YPBT2867), ail-like (YPBT2113), psaA and psaE. An internal fragment of the cpxR gene was used as a negative control. The approximate size of each amplified PCR fragment is given in parentheses. Where indicated, the purified CpxR-His tagged variants were incubated with ‘hot’ DNA templates at the concentrations of 0.75 and 1.5 µM. All reactions were performed in the presence of the phosphodonor acetyl∼P and binding specificity was aided by the constant presence of BSA and non-specific single stranded DNA. EMSAs were further controlled by a reaction containing 1.5 µM BSA instead of CpxR or competition reactions in which excess ‘cold’ DNA template competed with ‘hot’ DNA for available CpxR∼P. The electrophoretic mobility of the ‘hot’ DNA fragments in the absence of bound protein is highlighted by an arrow, while DNA-CpxR∼P complexes are signified with an asterisk (*). In some cases, the extent of mobility shifted DNA was dependent on the CpxR∼P concentration. Two asterisks (**) indicate residual non-specific background binding noise and are not believed to represent bona fide binding targets of CpxR∼P.