FIG. 3.

The SRI domain of Set2 binds synergistically to doubly modified CTD repeats. (A) Reverse far Western analysis. GST-yCTD and CTDK-I-phosphorylated GST-CTD (GST-yPCTD) fusion proteins were subjected to SDS-PAGE and transferred to nitrocellulose. Membranes were probed individually with purified recombinant full-length MBP-Set2 (Set2) or with MBP-SRI [Set2_(619-733)], and the bound MBP fusions were detected with an anti-MBP antibody. As a control, a duplicate membrane was probed with an anti-GST antibody (α-GST) to demonstrate the presence of both GST-CTD fusion proteins. (B) Increasing amounts of two MBP fusion proteins [Set2_(1-618) and Set2_(619-733)] were resolved in two SDS-polyacrylamide gels; one gel was subjected to far Western analysis with GST-[³²P]CTD as a probe, and the other was stained with Coomassie. (C) BIACORE analysis of the SRI domain. The MBP-SRI fusion protein [MBP-Set2_(619-733)] was analyzed by surface plasmon resonance (BIACORE) for binding to distinct phosphorylated synthetic three-repeat CTD peptides. These peptides were either Ser5 phosphorylated (5-phospho), Ser2 phosphorylated (2-phospho), or both (2 + 5-phospho) in each repeat (see Materials and Methods). Response units, on the y axis, represent binding to the peptides. The binding response to a scrambled control peptide carrying six SerPO₄residues (see Materials and Methods) has been subtracted from each of the three response curves. Only the peptide carrying both Ser2PO₄ and Ser5PO₄ in each repeat showed binding above control levels, and we estimate the affinity of this interaction (after subtraction of background binding to the control peptide) to be 6 μM.