FIG 2.
RpoE and RpoX directly bind to the rpoX promoter. (A) Diagram showing the promoter region of the rpoX gene. The RpoE- and RpoX-binding sites and the ribosome-binding site (RBS) are underlined. The regions protected by RpoE/RpoX are shadow boxed. The transcription start sites are labeled as +1 and marked in red. The red letters “TGA” and “ATG” are the stop codon of N646_4611 and the start codon of rpoX, respectively. (B and C) EMSA of RpoE and RpoX specifically binding to the rpoX promoter. Various concentrations of the RpoE and RpoX proteins were added to mixtures of poly(dI-dC) and Cy5-labeled rpoX promoter DNA (a 282-bp fragment of the promoter adjoining the start codon “ATG,” as indicated with the pair of arrows above the sequence). The same reactions were also carried out for a 300-bp fragment of the gyrB promoter region (negative control), which cannot be bound by RpoX and RpoE. (D) Western blotting of RpoX levels in WT, ΔrpoE, and ΔrpoX cells harboring pBAD33::PrpoX-Flag and grown at normal (30°C) or high (42°C) temperatures. RpoB was used as a loading control for the blots.