FIG. 4.

EMSA of MisR-misR promoter interaction. (A) A 608-bp PCR product (∼5 fmol) of a DNA fragment containing the misR promoter region (−504 to +104) was ³²P-labeled by T4 kinase, mixed with increasing amounts of MisR∼P (left) and MisR (right) for 20 min at 30°C and then subjected to gel electrophoresis. MisR∼P was generated by incubation with 50 mM acetyl phosphate for 30 min at 37°C. The amounts of MisR protein are 0, 34, 68, 136, 204, 272, and 340 pmol. (B) Competition EMSA. ³²P-labeled misR probe was mixed with the indicated amounts of either specific or nonspecific DNA, incubated with MisR∼P (102 pmol), and analyzed as described above.