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. 2009 Feb 10;37(6):1973–1983. doi: 10.1093/nar/gkp027

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© 2009 The Author(s)

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Context-independent DNA-binding by EbfC. (A) Interactions between EbfC and erpAB variant DNAs: wild-type b-WT without added protein (lane 1); probe b-C2, which contains a consensus sequence at site I and non-consensus sequences at sites II and III (lane 2); probe b-C20, which lacks consensus sequences at sites I, II or III (lane 3); probe b-C2+3, which lacks a consensus sequence at site II but contains consensus sequences at site I and III (lane 4); probe b-C1/2, which lacks consensus sequences at sites I, II or III (lane 5); and probe b-C1/2+3, which contains a consensus binding sequence at site III but lacks consensus sequences at site I and II (lane 6). All lanes contained 2 μM EbfC and 1 ng/μl poly-dI-dC. (B) Binding of EbfC to DNA probes derived from insertion of small DNAs containing consensus EbfC-binding sites into the TA-cloning site of pCR2.1. Lanes 1–3 contained b-SRK-A, a 34 bp insert containing two EbfC-binding sites. Lanes 4–6 contained b-SRK-B, which contains the same insert as does b-SRK-A except with mutations in both binding sites such that EbfC does not specifically interact (negative control). Lanes 7–9 contained b-SRK-C, which consists of a 12 bp insert containing one consensus EbfC-binding sequence. Lanes 10–12 contained b-SRK-D, which consists of a 7 bp insert with one consensus binding sequence. Lanes 1, 4, 7 and 10 lacked EbfC protein, lanes 2, 5, 8 and 11 contained 4 μM EbfC, while lanes 3, 6, 9 and 12 contained 12 μM EbfC. All lanes contained 1 ng/μl poly-dI-dC.