Regulation of the Escherichia coli HipBA Toxin-Antitoxin System by Proteolysis
Figure 7
HipB, HipB(W88A) and HipB72 bind hipBA operator DNA or HipA identically.
(A) Wild type HipB protein (red closed circle), HipB(W88A) protein (blue closed square) or the HipB72 C-terminal truncation protein (green closed triangle) was titrated into fluoresceinated hipBA O1O2 operator sequence and the change in fluorescence polarization (normalized millipolarization, mP) plotted as a function of the concentration of the titrant. The typical change in mP of each titration was between 60 and 80 units. The correlation coefficients for each curve fitting were 0.98, 0.99 and 0.99, respectively. (B) Wild type HipA protein was titrated into hipBA O1O2 DNA after the DNA was prebound by 20 nM wild type HipB monomer. Note that the concentration range is different in the left half and right half of the binding isotherms, with HipB titrations in the nanomolar range and HipA titrations in the micromolar range. (C) Wild type HipA protein was titrated into solutions containing 1 nM fluorescently labelled hipBA O1O2 DNA and titrated up to 50 nM wild type HipB monomer, 50 nM HipB(W88A) protein or HipB72 protein. This ensures stoichiometric binding of these HipB proteins to the DNA. Thus, the resulting binding affinity is formally between HipA and HipB that is bound specifically to hipBA DNA. The change in mP of each titration was between 88 and 150 units. The correlation coefficients for each curve fitting were 0.95, 0.99 and 0.99, respectively. A representative binding isotherm is shown for each protein binding to DNA or to the HipB-hipBA O1O2 complex.