Phosphorylation-dependent binding of EmbR to upstream regions of embCAB genes. (A) Gel mobility shift assays showing binding of EmbR to the promoter region of embCAB genes. An increasing amount of EmbR was incubated with 32P-labeled upstream regions of embC, embA, and embB genes at 4°C for 30 min. After incubation, EmbR-DNA complexes and free DNA were separated by nondenaturing polyacrylamide gel electrophoresis and subjected to autoradiography. The positions of EmbR-bound (solid arrows) and free (open arrows) probes are shown. (B) Phosphorylation of EmbR enhances its DNA binding activity. A total of 0.3 μg of unphosphorylated or phosphorylated EmbR (EmbR-P) was subjected to gel mobility shift analysis with 32P-labeled probes of upstream regions of embC, embA, and embB genes as indicated. The positions of EmbR-bound (solid arrow) and free (open arrow) probes are shown. Binding of phosphorylated EmbR with all three regions was examined in the presence of an excess amount (500×) of cold probe.