Figure 5.

VapC dimerization is required for conditional cooperativity. (A) Predicted tertiary structure of a VapC dimer showing positions of amino acid changes in the dimer interface. The predicted secondary structure of VapC is shown below the primary sequence. Amino acid patches involved in dimerization are shown in blue and red, and amino acid substitutions in these patches are indicated by vertical arrows. (B) LacZ activities of vapBC::lacZ transcriptional fusions carrying mutations in vapC. The genetic set-up of the transcriptional vapBC::lacZ fusion used is shown schematically below the diagram. A broken arrow pointing rightward indicates the vapBC promoter. TB28 (MG1655ΔlacZIYA) containing pKW254BC (vapBC::lacZYA) or its isogenic vapC substitution mutant derivatives (see Supplementary Table S1) were grown exponentially in LB medium at 37°C and specific LacZ activities were determined. (C) Gel shifts of vapO1 DNA with VapB and VapC (left panel) or VapC^Y72A (right panel). The proteins were mixed with radio labelled vapO1 DNA in given concentrations (nM) (lanes 1–7). U and C indicate positions of unbound and bond complexes, respectively. Right: Quantification of C band intensities (%) seen in (C) as a function of lane number in the gel-shift shown at left.