Figure 5
Binding in the control region of Sso0049 . A. Zoomed average ChIP-chip binding profile in the genomic region of Sso0049. Below the profile, the genomic environment is schematically depicted by representing ORFs as grey arrows. The region corresponding to the fragment tested in in vitro binding analyses, is boxed, whereas the identified Ss-LrpB binding sites are represented by vertical bars. B. EMSA of binding to a 176 bp fragment encompassing the Sso0049 control region. DNA populations are indicated as follows: free DNA (F), Ss-LrpB-DNA complexes (B1, B2 and B3) and complexed DNA retained in the wells of the acrylamide gel (W). Protein concentrations are given (nM). C. DNase I footprinting with Ss-LrpB binding to the Sso0049 control region fragment (having the top strand 32P labeled). A + G and C + T represent Maxam-Gilbert sequencing reactions whereas the other lanes contain DNase I-treated samples, with indication of the applied protein concentrations. Regions that are protected against DNase I upon addition of a low Ss-LrpB concentration (< 500 nM) are indicated with a vertical line to the left-hand side of the autoradiograph, while regions that are protected at high concentration are indicated on the right-hand side. Similarly, hyperreactive sites are indicated with ball-and-stick symbols. D. Sequence of the Sso0048/Sso0049 intergenic region with indication of the regions that are protected against DNase I in the experiment shown in subpanel C (dark grey = protected at all Ss-LrpB concentrations; light grey = only protected at high Ss-LrpB concentrations) and in an experiment using DNA with the bottom strand labeled. Coding sequences are shown in uppercase (left side = Sso0048, right side = Sso0049). The TSS of Sso0049, indicated with an arrow, was experimentally determined with primer extension analysis (Additional file 13: Figure S9).