Figure 2.

Purified RSC repositioned nucleosomes in salt gradient dialysis chromatin, but only in few cases, resulting in in vivo-like positions. DNaseI indirect end labelling analysis of the (A) PHO8, (B) RIM9, (C) CHA1 and (D) SNT1 promoter regions in vitro after assembly by salt gradient dialysis and incubation with WCE or purified RSC complex in the presence or the absence of ATP as indicated. The amount of RSC is given as the molar ratio of RSC to nucleosomes. In each panel, lanes 1 and 2 show the wt in vivo DNaseI pattern. Free DNA samples correspond to the respective non-assembled plasmids in the absence of WCE, RSC and ATP but under otherwise identical conditions. Bars in between lanes mark hypersensitive regions that correspond, at least to some degree, to NDRs of the in vivo patterns. The arrow between lanes 12 and 13 in D marks a nuclease-sensitive region that becomes inaccessible because of RSC activity. Ramps: increasing DNaseI concentrations. Position of marker bands is labelled relative to the ORF start of the respective locus. Schematics on the left are analogous to Figure 1A for the respective locus. Predicted Rsc3 binding sites (Supplementary Figure S8) are indicated by black dots.