RpoE binds directly to the <i>luxR</i> promoter region.

<p>(<b>A</b>) EMSAs were performed with purified RpoE, and the <i>luxR</i> promoter region was analyzed. The amount of RpoE protein (nM) that was used is indicated, and 20 ng of each Cy5-labelled probe was added to the EMSA reactions. The shifts were verified to be specific in experiments in which we added a 5- to 50-fold excess of unlabeled specific DNA and non-specific competitor DNA (poly(dI:dC)). (<b>B</b>) Plot showing the affinity of RpoE to the <i>luxR</i> promoter. The densitometric intensities of bound DNA fragments were plotted against RpoE concentrations. The arrow indicates the concentration of RpoE that caused half-maximal binding (<i>K</i>_d). (<b>C</b>)Footprinting analysis of RpoE binding to a binding site in the <i>luxR</i> promoter. Electropherograms of a DNase I digest of the P_<i>luxR</i> promoter probe (400 ng) after incubation with 0 or 400 nM RpoE. The respective nucleotide sequences that were protected by His-RpoE are indicated below, and the specific -10 and -35 regions are underlined. (<b>D-E</b>) <i>In vitro</i> transcription was performed using a P_<i>rpoE</i> template, (<b>D</b>) a P_<i>luxR</i> template (<b>E</b>), NTP, and RNAP core enzyme as well as RpoE. Various concentrations of purified LuxR were added into the reaction mixture to determine its effect on <i>luxR</i> transcription (<b>E</b>). The transcripts were purified, reverse-transcribed (RT, +) and detected using PCR. As a control, the same purified transcripts were also treated using the same process but without reverse transcriptase (RT, -).