Recruitment of TREX to the Transcription Machinery by Its Direct Binding to the Phospho-CTD of RNA Polymerase II
Figure 5
THO binds directly to the S2/S5 diphosphorylated CTD.
Pulldown experiments were performed with immobilized CTD peptides that were not phosphorylated (0), mono-phosphorylated on tyrosine 1 (Y1P), serine 2 (S2P) or serine 5 (S5P), diphosphorylated on Y1/S2 (Y1PS2P), Y1/S5 (Y1PS5P) or S2/S5 (S2PS5P) or S2PS5P dephosphorylated by treatment with alkaline phosphatase (S2PS5P+AP). The THO complex binds to CTD peptides phosphorylated on S2 (S2P) and S5 (S5P) and more strongly to the S2/S5 diphosphorylated CTD (S2PS5P). Binding of THO to the CTD is dependent on S2/S5 diphosphorylation since treatment with alkaline phosphatase (AP) of the S2/S5 diphosphorylated CTD peptide abrogates binding of THO (S2PS5P+AP). The unrelated Rix1 complex served as negative control. Pcf11 was used as a positive control for association with the S2 and S2/S5 (di)phosphorylated CTD. The TAP-tagged protein of each complex was detected by Western blotting against CBP (Hpr1 for THO, Rix1 for the Rix1 complex and Pcf11 for the Pcf11 complex). A representative experiment is shown.