FIG. 5.

In vitro transcription analysis of different phyC fragments. The transcription reactions were carried out in 20-μl volumes in transcription buffer by use of 200 fmol of the template, 0.25 μM PhoP∼P, and 0.1 μM RNA polymerase (B. subtilis). The F3 fragment, harboring the entire phyC promoter region with AbrB binding sites (A), and the F4 fragment, harboring the promoter proximal AbrB binding site 2 (B), were transcribed in the presence of various AbrB or AbrBQ82K concentrations as described for Fig. 4. The concentration of 1 μM AbrB or AbrBQ82K is indicated by an asterisk. (C) In vitro transcription of F4 in the presence of 1 μM AbrB and increasing concentrations of the complementary F5 DNA fragment (Fig. 1). (D) In vitro transcription of fragment F5, which does not contain the phyC coding region, in the presence of 1 μM AbrB or AbrBQ82K and increasing concentrations of the complementary fragment F4. The DNA fragments were added at amounts from 0 fmol to 400 fmol in 50-fmol steps.