Figure 3

Electrophoretic mobility shift assays using purified CysR to identify the co-activators of the protein (A) and test for specific binding (B+C). CysR was purified using the IMPACT-C system (New England Biolabs) in presence of 0.1% Triton X-100. A Cy3-labelled 523 bp fragment containing the cysI - fpr2 intergenic region was incubated with a 1,000 times molar excess of the purified protein in presence of different intermediates of sulphur metabolism (OAS, O-acetyl-L-serine; NAS, N-acetyl-L-serine; OAH, O-acetyl-L-homoserine) at 10 mM concentration, demonstrating that CysR can only bind to DNA in presence of either OAS or OAH (A). Tests with OAS-activated CysR and either a negative control (123 bp internal fragment of cg2118, B) or herring sperm blocking DNA (C) revealed that activated CysR binds non-specifically in vitro.