Figure 4. Both ArnR boxes are required to facilitate ArnR and ArnR1 binding to pflaB.
(A) flaB (upper) promoter sequence. Numbers correspond to the distance respective to the transcription start site (TSS). The position of core promoter elements and cis-regulatory sequences is depicted in boxes (Lassak et al., 2013) BRE: factor B recognition element, TATA: TATA-box. pflaB41 (black box) comprises the region of −43 to −84 and includes both ArnR boxes. The 15 bp regulatory elements ArnR box-1 and box-2 are highlighted in green and fragments used in this assay covering the sequence of either box are circled by dashed lines. (B) Net fluorescence obtained in microscale thermophoresis (MST) assays with increasing amounts of ArnR (left graph) and ArnR1 (right graph). 0.156–18 µM ArnR or ArnR1 were titrated in 2.5 nM Alexa Fluor 647 labeled DNA and subjected to thermophoresis. Curves were obtained from two independent experiments performed in duplicate. Fluorescence was normalized (ΔF) by subtracting the lowest measured fluorescence value determined within each experiment from all measured fluorescence values. Obtained curves were fitted using non-linear regression, with one site specific binding according to (y = Fmax*X/(KD + X).