Fig. 3: The impact of CTD heptad residues on phosphorylation by CDK7 and CTD phosphorylation analysis under phase-separation conditions.

a The initial reaction velocities of the human CDK7 complex with the mGFP-CTD substrate and its variants relative to the wild-type CTD. Black dots indicate the individual measurements (n = 3). Bars, mean; error bars, ± SD. b Representative micrographs of three independent in vitro LLPS kinase assays with 5 μM mGFP-CTD (green) and 0.5 mM ATP after 5 and 10 min with indicated concentrations of the CDK7 complex. Scale bar = 10 μm. c Quantifications of experiments shown in (b). Each dot represents a detected droplet, black dots indicate median of droplet size per measurement (n = 3). The black line and error bar represent the mean ± SD of the three medians. d Representative snapshots from CG condensed phase simulations of CTD molecules phosphorylated at S5. Rendering same as (b) in Fig. 2. e The CDK7 complex sedimentation assay in the presence of mGFP-CTD. A scan of representative SDS-PAGE (top)—(M) molecular weight marker; (I) input; (S) supernatant; (P) pellet. Quantifications of the sedimentation assays (bottom) (n = 3), mean ± SD. f Representative micrographs of at least two independent in vitro LLPS assays with mGFP-CTD (green) and increasing concentration of the CDK7 complex labeled with Alexa594 (red). Merge = overlay of GFP and Alexa 594 channels. Scale bar = 1 μm. (*) The intensity of all micrographs was uniformly enhanced in Fiji¹²⁵ for better visibility. g Super-resolution micrographs show the CDK7 complex (red) localization at the surface of the mGFP-CTD (green) droplets. h Kinase assays with mGFP-CTD at different concentrations and 0.5 mM ATP with the CDK7 complex (at 5 nM), in the presence and absence of 10% dextran (n = 3), respectively, mean ± SD. i (left) Schematic representation of the sedimentation kinase assay workflow. i (right) Quantification of the sedimentation kinase assay (n = 3), mean ± SD. Statistical significance for (e, h, i) was determined by unpaired, two-sided t-test. p-values: (e) * = 0.030, (h) ** = 0.0093, * = 0.0292, **** ≤ 0.0001, (i) ** = 0.0012, **** = 0.0002. Source data are provided as a Source Data file.