FIG. 1.

Identification of a novel region in Set2 required for RNAPII binding. (A) Schematic representation of the Set2 constructs used to probe for RNAPII interaction. The SET domain along with the AWS domain, postSET domain (PS), WW domain, and coiled-coil motif (CC) are shown. All constructs contained a C-terminal Flag epitope. (B) set2Δ cells were transformed with either vector only or plasmids coding for the indicated Set2-Flag construct, and WCE were prepared. WCE were immunoprecipitated with anti-Flag beads followed by immunoblotting with antibodies directed against serine 5-phosphorylated CTD (H14; α-Ser5P), serine 2-phosphorylated CTD (H5; α-Ser2P), or the Flag epitope. Significant to mention is that the H5 antibody may also recognize serine 5 CTD phosphorylation in addition to serine 2 phosphorylation (16). Asterisks indicate the location of nonspecific Flag antibody-reactive species. (C) Schematic representation of the Set2-SRI domain constructs used to determine the boundaries of the functional SRI domain. N- and C-terminal truncations of the SRI domain were made in 15-amino-acid increments as shown. All constructs contained a C-terminal Flag epitope. (D) set2Δ cells were transformed with the indicated plasmids, WCE were prepared, and co-IPs were performed using the antibodies indicated in panel B. Sizes of the molecular mass markers are shown.