FIG. 5.

War1p binds to the WARE in the PDR12 promoter. (A) For mobility shift experiments, the ³²P-end-labeled WARE probe (bp −655 to −604 of the PDR12 promoter) was incubated with GST-War1p alone (lanes 2 and 3) or in the presence of a specific (S; lanes 5 and 6) or unspecific (US; lanes 6 and 7) competitor probe for 30 min. WARE alone (lane 1) or extracts from uninduced E. coli (lanes 8 and 9) served as controls. Complexes were separated by native gel electrophoresis and detected by autoradiography. (B) For in vivo footprint experiments, FY1679-28c (lanes 1 and 2) or YAK100 (Δwar1; lanes 3 and 4) cells were grown to the logarithmic growth phase and stressed with 8 mM sorbate for 10 min (+; lanes 1 and 3) or left unstressed (−; lanes 2 and 4). Cells were treated with DMS to methylate DNA. Primer extension was carried out on both strands (left, antisense; right, sense) to detect DNA methylation. Products were separated by native acrylamide gel electrophoresis and autoradiographed. Graphs reflect intensities of the bands (lane 1, solid line; lanes 2 and 3, dashed line). A, B, and C, sequences in the WARE. Boxed nucleotides show changes in methylation intensity corresponding to protection or deprotection.