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. 2011 Aug 18;6(8):e23314. doi: 10.1371/journal.pone.0023314

Figure 4. Mapping the CpxR∼P DNA binding site upstream of rovA by nuclease protection (footprinting) analysis.

Figure 4

DNase I footprinting assays were performed to investigate the binding of CpxR∼P to a region within the rovA, ppiA and cpxR-cpxP promoters. These 314 base pair (bp), 276 bp and 245 bp fragments respectively, were labeled on the sense strand before being incubated with CpxR∼P at the following final concentrations: 50 nM, lane c; 100 nM, lane d; 200 nM, lane e; 400 nM, lane f. The absence of CpxR∼P in lanes a and b is indicated by ‘–’. Reactions were resolved by denaturing PAGE and analyzed with a Molecular Dynamics PhosphorImager. Labeled pBR322 DNA digested with MspI (New England Biolabs) was used as a size marker (lane a). An estimation of the protected sequence is given on the right hand side of the panels. Based upon the E. coli consensus sequence of 5′-GTAAA(N)4–8GTAAA-3, a putative CpxR∼P consensus binding site is highlighted in a gray box. A labeled internal fragment (389 bp) within the cpxR open reading frame served as a non-protected control.