Fig. 3.

SinR binding to DNA. (a) The arrangement of SinR binding motifs at the B. subtilis eps, yqxM and aprE promoters. SinR binding sites are shown as boxes, with the putative SinR binding sequence displayed. The relative orientation of the sequences is shown by the arrows. Numbers between boxes indicate the number of base pairs between the sequences. Numbers below the boxes represent their position with respect to the transcription start site at + 1, which is indicated by an arrow. (b) Oligonucleotide sequences used for DNA binding studies in this work. The double-stranded oligonucleotide duplexes contain the 7-bp SinR binding motifs, highlighted in bold, in three arrangements/orientations. (c) Fluorescence anisotropy measurement of SinR binding to 22-bp oligonucleotide duplexes. The binding curves show an increase in the anisotropy of the fluorescence of the labelled DNA upon successive addition of SinR. The calculated anisotropy values (squares) were fitted to a model (line) using the Scientist software. The lines in purple represent the inverted repeat, the lines in blue represent the tandem repeat and the lines in red represent the single-site duplex. SinR binds with 10-fold higher affinity to the inverted repeat sequence and with similar lower affinity to the tandem repeat and single-site sequences. (d) SPR analysis of SinR binding to the inverted repeat DNA duplex. The curves show an increase in response units (RU) as SinR binds to DNA, followed by a decrease as the complex dissociates. The SinR injection began at 80 s and ended at 320 s. Each sensorgram is a binding experiment carried out at a different SinR concentration in the range 0–3000 nM. Data from the SPR sensorgrams were used to plot a steady-state binding curve for the interaction of SinR with the inverted repeat oligonucleotide. The point corresponding to 0 nM SinR was set to 0 RU, and all other data points were scaled to this. The point at 0 nM SinR and 0 RU is omitted.