Acetylation-Dependent Binding Analysis of the Yeast Gcn5 Bromodomain Protein

The 439 amino acid yeast Gcn5 protein contains a C-terminal bromodomain, which is required for SAGA (Spt-Ada-Gcn5-Acetyltransferase) mediated nucleosomal acetylation and transcriptional coactivation. Bromodomains are acetyl-lysine binding modules found in many chromatin binding proteins and histone acetyltransferases. Recently, both in vivo and in vitro studies indicate that bromodomains are able to discriminate the acetylation state of lysine side-chains within histone proteins. Here, the cloning, expression and bioactivity of a recombinant bromodomain from the yeast Gcn5 protein is described. The bromodomain from Gcn5 was cloned from yeast genomic DNA enabling effective one-step purification by affinity chromatography. Steady-state fluorescence anisotropy was used to quantify the interaction of Gcn5 with acetylated histone H3. The present cloning, expression, and purification procedure enabled the preparation of large quantity and high yields of biologically active recombinant Gcn5 bromodomain for in vitro structure and function studies.