FIG. 2.

Recombinant Esa1p acetylates histones. Products of HAT assays using either yeast or calf histones as substrates were resolved on an SDS–18% polyacrylamide gel that was stained with Coomassie brilliant blue (A) and treated for fluorography (B). The activities of recombinant Esa1p were compared with that of recombinant Gcn5p; whereas Esa1p preferentially acetylates H4, Gcn5p preferentially acetylates H3 (32). (C) Bacterial extracts containing equivalent amounts of recombinant Esa1p, vector control, or recombinant Esa1-L327Sp were incubated with ³H-acetyl coenzyme A and either calf histones or BSA. Esa1p acetylates calf histones but not BSA. No activity was observed with vector control or Esa1-L327Sp lysates or with two other mutant proteins tested (data not shown). The preferred targets of Esa1p are Lys5 of histone H4 (D), Lys14 of histone H3 (E), and Lys4 and Lys7 of histone H2A (F). Note that lysines 8, 12, and 16 in H4 were also acetylated. When repetitive yields for the cycles of Edman degradation were calculated, the extent of acetylation at each of these sites was equivalent and significantly less than that of Lys5.