Extended Data Fig. 8 |. CYCT1 binds directly to the Pol II CTD in an HRD-dependent manner and the binding is enhanced after the CTD is phosphorylated by CAK (CDK7–CYCH–MAT1).

a, Immobilized GST–CTD was incubated with recombinant GFP–T1-IDR or GFP–T1-IDRΔHRD. The input (2.5%) and the bound proteins were analysed by western blotting. b, Binding reactions containing the purified recombinant fusion proteins indicated on the right were analysed in a 7.5 to 20% glycerol gradient containing 500 mM NaCl plus 0.5% NP-40, which was centrifuged at 55,000 r.p.m. and 4 °C for 13 h. The indicated fractions were analysed by western blotting to detect the distributions of proteins marked on the left. The entire length of the CTD could be bound by varying numbers of IDRs, resulting in the formation of a series of complexes with broad distributions in the gradient. c, mCherry–CTD was incubated with or without immobilized CAK for 6 h in kinase reactions and then analysed by SDS–PAGE and Coomassie blue staining. d, Immobilized GST–CTD was incubated with (phos.) or without (unphos.) CAK for 6 h in kinase reactions. After washing, the GST–CTD beads were incubated with GFP– T1-IDR. The indicated proteins were eluted off the beads and analysed by western blotting.