Figure 5

(a) Interaction of Mediator with RNAPII in the presence of wild-type, ΔMed13, or ΔCdk8 CKM. Under this assay conditions, wild-type CKM blocks interaction of RNAPII with Mediator, but ΔMed13 CKM or ΔCdk8 CKM have no effect. (b) Interaction of GST-CTD with Mediator in the presence of wild-type, ΔMed13 or ΔCdk8 CKM. The effect of the different CKMs on CTD interaction with Mediator mirrors their effect on RNAPII interaction. (c) Comparison between 3D EM maps of Mediator–RNAPII and Mediator–CKM complexes highlights the overlap between RNAPII and CKM densities that results from binding of both to the same portion of the Mediator structure. (d) Silver-stained SDS-PAGE analysis of purified Srb10-1 CKM comprising Med13, Med12, CycC, and a C-terminal truncated Cdk8 (Cdk8-Cd) kinase (left). A class average calculated from images of Srb10-1 CKM particles shows that the CKM complex appears intact (the C-terminal deletion in Cdk8 is too small to be apparent at the resolution of the class average). (e) Effect of the Srb10-1 Cdk8 C-terminal truncation on Mediator-CKM interaction. Nearly 60% of Mediator-CKM particles formed with wild-type CKM show the Cdk8-Middle module contact. In contrast, only ~10% of Mediator-CKM particles formed with Srb10-1 CKM show a contact with the Middle module. (f) Silver-stained SDS-PAGE analysis of purified Srb8-1 CKM shows that it only contains CycC and the Cdk8 kinase. All scale bars represent 10 nm.