Figure 4

Hydroxyl radical footprinting of the top (IRRA) and bottom (IRRB) strands of the IS 2 IRR. (A) Quantitative analysis panel, with tracings (derived from the color-coded gels immediately below the panel) showing relative intensities of bands from the footprinted, cleaved, bound top strand of IRR, (IRRA-red tracing) and the control, cleaved, unbound, free DNA, (green tracing). The protection profile is shown as horizontal bars within the panel identifying troughs of weakly (grey) and strongly (black) protected residues that are significantly below the green control. Determination of strong and weak protection was based on the combined analysis of visual evidence of a band and the absence or presence of peaks within the troughs. Visual absence of a band coupled with absence, or only a suggestion, of a peak defined strong protection. A faint band which showed a small peak within a trough defined weak protection. Bands and peaks are numbered (1 to 41) from the outer (3') end of IRRA to the inner end. Individual peaks are identified by dots and numbered vertical lines identify the nature of every fifth base. Asterisks identify enhanced residues whose red peaks rise significantly above those of the green control. The sequence of IRRA, shown below the panel was used to annotate the peaks in the upper panel and the bands in the color coded lanes. Nucleotides are numbered as described above. The IRR sequence within the large brackets, is flanked by host DNA at the outer (3') end of the terminus (-1 to -9) and the sequence of IS2 adjacent to the inner end of the terminus (42 to 45). (B) Quantitative analysis panel showing relative intensities of bands from the footprinted IRRB DNA (red) and the control DNA (green) derived from the gels shown immediately below the panel as described in part (a). IRR: right inverted repeat.