The phosphorylation state of the C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II changes as polymerase transcribes a gene, and the distinct forms of the phospho-CTD (PCTD) recruit different nuclear factors to elongating polymerase. The Set2 histone methyltransferase from yeast was recently shown to bind the PCTD of elongating RNA polymerase II by means of a novel domain termed the Set2-Rpb1 interacting (SRI) domain. Here, we report the solution structure of the SRI domain in human Set2 (hSRI domain), which adopts a left-turned three-helix bundle distinctly different from other structurally characterized PCTDinteracting domains. NMR titration experiments mapped the binding surface of the hSRI domain to helices 1 and 2, and Biacore binding studies showed that the domain binds preferably to [Ser-2 ؉ Ser-5]-phosphorylated CTD peptides containing two or more heptad repeats. Point-mutagenesis studies identified five residues critical for PCTD binding. In view of the differential effects of these point mutations on binding to different CTD phosphopeptides, we propose a model for the hSRI domain interaction with the PCTD.
RNA polymerase II carries an intrinsically unstructured, flexible domain at the C terminus of its largest subunit. The principal function of this C-terminal repeat domain (CTD), which comprises multiple repeats of a consensus heptamer Y 1 S 2 P 3 T 4 S 5 P 6 S 7 , is to serve as a binding scaffold for various nuclear factors (reviewed in refs. 1-3). The CTD of preinitiating RNA polymerase II is mostly unphosphorylated, whereas after initiation and during elongation it is hyperphosphorylated, principally on Ser-5 and Ser-2 residues of the repeats (4-6); we refer to this form as the phospho-CTD (PCTD). Attendant with changes in patterns of CTD phosphorylation, the ensemble of CTD-bound proteins changes as RNA polymerase II progresses through the transcription cycle (5). Although knowledge of the number and types of PCTD-associating proteins (PCAPs) has expanded rapidly (7, 8), information about the molecular nature of PCAP-PCTD interactions remains quite limited; detailed binding properties and͞or 3D structures are known for only a few PCTD-interacting domains (PCIDs) (9-14).Describing in molecular detail the interactions between the PCTD and its binding partners is an important step in advancing our understanding of the PCTD and its manifold functions. One recently identified binding partner of the PCTD is the Saccharomyces cerevisiae Set2 (yeast Set2; ySet2), a histone methyltransferase that modifies K36 of histone H3 in nucleosomes of transcribed genes (15)(16)(17)(18)(19). The identification of ySet2 as a PCAP together with studies of transcription in set2 mutant strains suggests a role for the PCTD in chromatin structure modulation during elongation (20). Deletion studies of ySet2 mapped a novel PCID, termed the Set2-Rpb1 interacting (SRI) domain by Kizer et al. (20), to the C-terminal segment of ySet2. The yeast SRI domain binds preferentially to PCTD peptides with both S...