FIG. 2.

Probing the interaction between Rgt1 and its binding sites. (A) Footprinting assays using DNase I, DMS, and UV as probes were carried out with a DNA fragment of the HXT3 promoter containing five Rgt1 binding sites (−372 to −572 [Fig. 3A]) as described in Materials and Methods. For DNase I and DMS protections, a ³²P-labeled DNA fragment (2 × 10⁴ cpm) was incubated with different amounts of Rgt1 (30, 60, and 120 ng, from the third gel lane from the left). The first (L) and second gel lanes are the A+G ladder and the control without Rgt1, respectively. For UV photofootprinting, Rgt1 (100 ng) was incubated (+) with the same amounts of DNA used for DNase I and DMS protections, and then DNA-protein complexes were irradiated with UV for the time indicated above each lane. The Rgt1 binding sites are indicated by boxes. (B) Summary of interactions between Rgt1 and its binding sites observed from three different footprinting assays. DMS protection assays show that Rgt1 weakly contacts CGG at C₁ and C₂ (open circles).