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. 2019 Jul 12;10:1563. doi: 10.3389/fmicb.2019.01563

Figure 5.

Figure 5

DNAse I and in gel copper-phenanthroline (OP-Cu) footprinting of Lrp binding to the artP control region (top or coding strand revealed). (A) EMSA with Lrp binding to the artP control region used for the in gel footprinting with the [(OP)2-Cu+] ion. Lrp concentrations (in μM) are expressed in monomer equivalents. The position of free DNA (F), single stranded DNA (ss) and the various complexes with a different migration velocity (B1 to B4) are indicated. (B) Autoradiograph of in gel footprinting of free DNA (F) and the major Lrp-DNA complexes B3 and B4 with a different migration velocity extracted from the gel presented in panel (A) after treatment with the [(OP)2-Cu+] ion and separation of the reaction products by gel electrophoresis in denaturing conditions. Regions of protection in the different complexes are indicated with gray and black colored lines for the complexes B3 and B4, respectively. A + G and C + T represent the Maxam-Gilbert sequencing ladders. In DNase I footprinting the black triangle on top of the autoradiograph represents increasing Lrp concentrations corresponding to 0, 0.46, 0.92, 1.86, 3.68, and 7.36 μM. Regions protected against DNase I cleavage are indicated with a blue colored line. The short lightblue colored line represents the region that is already protected at the lower Lrp concentrations (1.86 μM, nucleation site), the long blue colored line represents the extended zone of protection observed at higher Lrp concentrations. A short horizontal bar with a dot represents a site that becomes hyperreactive to DNase I cleavage upon Lrp binding. (C) Sequence of the artP control region (coding strand) with indication of regions of protection against enzymatic and chemical cleavage. The region of protection in complex B3 is gray shaded. Other symbols are as in panel B. The −10 and − 35 promoter elements are underlined. A short arrow represents the start of transcription (+1). The in silico predicted Lrp binding site (−85 to −98) (Cho et al., 2008) is boxed. Red colored letters represent ARG box sequences.