FIG. 4.

DNA footprint of NadR on nadA operator. Basic conditions for the assay performed by using His-tagged NadR are the same as described in the legend to Fig. 3B. Lanes marked G, A, T, and C are a DNA sequencing ladder of the coding strand (A) and template strand (B) of the 225-bp nadA operator-containing PCR fragment (pFP132). Lanes 1 to 4 show the results of DNase I protection assays. For panel A, lanes 1 and 2 contain 22 nM His-tagged NadR, and lanes 3 and 4 contain 11 nM His-tagged NadR. Lanes 1 and 3 contain no NAD. Lanes 2 and 4 contain 660 μM NAD. For panel B, lane 1 contains no NadR while lanes 2 and 3 contain 11 nM His-tagged NadR. Only lane 3 has 660 μM NAD. The DNA sequence of the nadA operator is shown to the left in each panel. Consensus −10 and −35 RNA polymerase recognition sequences are indicated. Bases protected against and hypersensitive to DNase I treatment are illustrated adjacent to the printed sequence by boxes and circles, respectively. A rectangle next to the footprint indicates the location of the protected region. Brackets around printed sequence indicate bases consistent with consensus NAD box sequences (TGTTTA and inverted repeat).