Fig. 5.

Promoter occupancy by Ptr2 and histones. A 460 bp DNA probe encompassing positions -302 to +158 relative to the start site of transcription of the rb2_con promoter, 5^′-end labeled on the top (nontranscribed) strand (A) or the bottom (transcribed/template) strand (B), was generated by PCR using plasmid pTerm as template. This probe was incubated at 65 °C for 20 min in the absence of HMjA1 (Lane 1 of each panel) or with increasing concentrations of HMjA1 (indicated above each panel), and subjected to hydroxyl radical (•OH) cleavage for 30 s at the same temperature. In lanes 6 to 9, HMjA1 binding to DNA is analyzed in reaction mixtures also containing 100 nM His₆-Ptr2. In each panel, lane 10 shows His₆-Ptr2 binding to DNA in the absence of histone. The TATA box and the two Ptr2 consensus binding sites are identified at the left of each panel. Also shown are the untreated DNA probe (P) as well as the A + G chemical sequencing ladder. The bullets point to the shift of protection at the Site 2 dyad reflecting histone displacement by Ptr2. (C) Aligned density profiles of •OH footprints shown in panel A; profiles in black and red correspond to lanes 1 and 2, respectively. The horizontal bars indicate the two consensus Ptr2 binding sites, with the arrows pointing to their central base pairs, which are the most protected by Ptr2 against •OH cleavage.