Figure 7.—

In vitro HAT activity of recombinant Esa1. The wild-type and catalytic-site mutants were N-terminally tagged with a His₆ epitope tag and expressed in E. coli. After purification on Ni-NTA resin, relatively equal amounts (top, Esa1 CBB; note that the Esa1-E338Q samples have slightly more protein present) of Esa1 (or no Esa1 as a control) were combined with chicken core histones and [³H]-acetyl CoA at either pH 8.0 or pH 9.2 and incubated at room temperature for 30 min. To assay incorporation of [³H]-acetyl groups into the histones, we ran the reaction on SDS–PAGE, dried the gel, and subjected it to fluorography (middle). A Coomassie stain of the same region of the gel is shown to confirm equal loading of histones (bottom).