FIG. 4.

Autoradiograph of EMSA results, showing the binding ability of RepB(His)₆ protein [present in crude protein extract of RepB(His)₆-overproducing E. coli M15 or purified] in ³²P-labeled inc1 and inc2 regions of pTAV320. (A and B) inc1 fragment (A) and inc2 fragment (B) incubated with different concentrations (2.5, 5, 10, and 20 μg/ml in lanes 2 to 5, respectively) of M15(pQE30/repB) crude protein extract. Lane 1 contained labeled fragment with no protein added. (C) inc1 fragment (lanes 1 to 3) and inc2 fragment (lanes 4 to 6) incubated (i) with M15(pQE30) crude protein extract (20 μg/ml) (lanes 2 and 5), (ii) with purified IHF protein (3 μg/ml) (lanes 3 and 6), and (iii) without protein (lanes 1 and 4). (D) The inc2 fragment was digested with PvuII (lanes 1 to 3) and incubated with 2.5 and 5 μg of purified RepB(His)₆ protein per ml (lanes 2 and 3, respectively). No protein was added to the reaction mixture in lane 1. The 126-bp PCR-amplified fragment of the inc2 region (Fig. 2B) (lanes 4 to 6) was incubated with 2.5 and 5 μg of purified RepB(His)₆ protein per ml (lanes 5 and 6, respectively). No protein was added to the reaction mixture in lane 4. (E) An 18-bp oligonucleotide containing a single R1/R2 sequence was incubated with 0.5, 1, 2.5, 5, and 10 μg of purified RepB(His)₆ protein per ml (lanes 2 to 6, respectively). No protein was added to the reaction mixture in lane 1. The positions of the protein-DNA complexes and the sizes of the unbound probes are indicated on the right and left. ssDNA, single-stranded DNA.