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. 2010 Apr 26;38(16):5443–5455. doi: 10.1093/nar/gkq284

Figure 5.

Figure 5.

Identification of the high-affinity BpaB-binding site in erp Operator 2, by use of simultaneous titration of multiple probes with increasing concentrations of BpaB321. In this method, several DNAs that overlap in sequence were simultaneously incubated with protein, then subjected to EMSA. These DNAs which contain the high-affinity protein binding site were preferentially bound by lower concentrations of protein, and signals corresponding with those free DNA disappeared soonest. (A) Sequences of labeled DNA probes used in simultaneous EMSAs. Asterisks indicate DNAs preferentially bound by BpaB. The –35 sequence of the erpAB promoter and the two consensus EbfC-binding sites are indicated by underlining. The upper seven nested DNAs were anchored 51 bases downstream of the erpAB transcription start site, and EMSA data are illustrated in (B). The lower seven nested DNAs were anchored at bp –174 relative to the start of erpAB transcription, and EMSA data are illustrated in (C). The sequence contained in all DNAs that preferentially bound BpaB but absent from lower-affinity DNAs is indicated by a thick horizontal bar. (B) Simultaneous EMSA of recombinant BpaB321 with the upper seven DNAs of (A). Lane 1 contains only the DNAs. Lanes 2–12 contain DNAs plus increasing concentrations of recombinant BpaB321. Quantification of signal strengths indicated that BpaB bound preferentially to the largest DNA (marked with an arrowhead): in lanes 9, 10 and 12, the proportion of free DNA for this probe was 21, 0.10 and 0% of lane 1, respectively, while that of the next largest probe was 52, 13 and 10% of lane 1, respectively. (C) Simultaneous EMSA of recombinant BpaB321 with the lower seven DNAs of (A). Lane 1 contains only the DNAs. Lanes 2–12 contain DNAs plus increasing concentrations of recombinant BpaB321. Quantification of signal strengths indicated that BpaB bound preferentially to the five largest DNAs (marked with arrowheads): in lanes 7, 8 and 9, the proportion of free DNA for the largest probe was 16, 10 and 0% of lane 1, respectively, while that of the smallest probe was 75, 55 and 43% of lane 1, respectively.