FIG. 3.

TR-dependent recruitment of CDK8-Mediator and Pol II into a functional PIC at the DioI promoter in vitro. (A) Schematic representation of the DioI promoter, showing the locations of TREs and biotin-conjugated PCR primers. (B) Single-round immobilized PIC assembly and transcriptional initiation assay. (C) TR-dependent recruitment of CDK8-Mediator and Pol II. Biotinylated immobilized DioI template was incubated with or without purified baculovirus-expressed TRα, RXRα, or T3 (10⁻⁷ M) in HeLa cell nuclear extract for 40 min. The PICs were then isolated by using streptavidin beads, washed, fractionated by SDS-PAGE, and then probed by immunoblotting using the specific antibodies indicated to the right of each panel. (D and E) CDK8 and Pol II dissociate from the PIC upon transcription initiation. PIC assembly on immobilized DioI promoter templates was carried out as described for panel C. The PICs were then isolated by using streptavidin beads, washed, and then resuspended in transcription buffer containing NTPs (100 μM) for the times indicated (D) or containing ATP or NTPs (100 μM) for 2 min (E). The streptavidin conjugates were then precipitated, washed, fractionated by SDS-PAGE, and then probed by immunoblotting using the specific antibodies indicated to the right of each panel.