FIG. 2.

Gel retardation analysis of LmrA binding to the lmr promoter region. (A) Probe settings. The positions and orientations of the PCR primers used for probe preparation are indicated schematically. The pair of horizontal arrows facing each other indicates the incomplete palindrome sequence. The probe designations are indicated on the left, and the thick horizontal lines and dotted lines indicate the stretches present and deleted in each of the probes, respectively. On the right, the results of gel retardation assay are summarized (Yes and No indicate LmrA binding and no LmrA binding, respectively). (B) Interaction between LmrA and the MK1-MK2 probe. The MK1-MK2 probe (0.02 pmol) was mixed with a protein extract of JM109 cells carrying plasmid pLMRA to obtain a reaction mixture (25 μl) (lane 2, 7.5 μg; lane 3, 3.8 μg; lane 4, 1.9 μg; lane 5, 0.9 μg) or pUC18 (lane 6, 7.5 μg) grown in the presence of 1 mM IPTG, and without the extract (lane 1). The positions of LmrA-probe complexes (bound) and free probe (free) are indicated on the right. (C) Deletion analysis. Gel retardation experiments were performed like the experiments described above. Each of the probes indicated was mixed with an extract of JM109 cells carrying pLMRA (lanes 2, 7.5 μg; lanes 3, 3.8 μg) or pUC18 (lanes 4, 7.5 μg) or with no extract (lanes 1). The position of LmrA-probe complexes is indicated by arrows.