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. 2012 Mar;194(6):1523–1532. doi: 10.1128/JB.06104-11

Fig 5.

Fig 5

Analysis of dinJ-yafQ promoter region and its interaction with DinJ-YafQ(His)6 protein complex. (A) E. coli intergenic region containing dinJ-yafQ promoter and palindromic sequences. The important predicted (BPROM) promoter sequences of dinJ and yafL genes are boxed, the putative LexA binding site is indicated in boldface, broken arrows indicate palindromic sequences, and letters in italics indicate the palindrome sequence regions. (B) EMSA analysis of interaction of DinJ-YafQ(His)6 complex with 376-bp dinJ-yafQ promoter DNA fragment. A total of 8 pmol of PCR-amplified dinJ-yafQ promoter DNA fragments was incubated in 10 μl of binding buffer (10 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, 5 mM MgCl2, 2.5% glycerol [pH 7.5]) with increasing amounts of protein at 22°C for 30 min. The EMSA was visualized in a 6% polyacrylamide gel stained with ethidium bromide. Lanes 1 to 5, promoter DNA incubated with 0, 0.5, 1, 2, and 4 pmol of DinJ-YafQ(His)6 protein complex; lanes 6 to 9, promoter DNA with 10, 20, 40, and 80 pmol of DinJ(His)6, lanes 10 to 13, promoter DNA with 10, 20, 40, and 80 pmol of (His)6DinJ. (C) EMSA analysis of DinJ-YafQ(His)6 protein complex interaction with the palindromic sequences of dinJ-yafQ promoter region. EMSA was performed to detect the interactions of purified DinJ-YafQ(His)6 protein complex with three 26-nucleotide regions of the dinJ-yafQ promoter, containing palindrome I (Pal I), 3′ part of palindrome I and the 5′ part of palindrome II (Pal I-II), and palindrome II (Pal II). Random DNA sequence was used as a control (control DNA). A total of 12.5 pmol of each DNA fragment (prepared as described in Materials and Methods) was incubated in 10 μl of binding buffer (10 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, 5 mM MgCl2, 2.5% glycerol [pH 7.5]) with increasing amounts of protein (0, 8, 15, and 30 nmol) at 22°C for 30 min. EMSA results were visualized by ethidium bromide staining of an 8% polyacrylamide gel. The open arrowhead indicates free DNA; full arrowheads indicate DNA bound in complex with DinJ-YafQ(His)6.