Figure 4.

Dissolution of RNA pol II CTD condensates requires high concentration of 1,6-hexanediol rendering Cdk7 inactive.A, solution of 10 μM full-length human GST-CTD_[52] in aqueous buffer (left). After addition of dextran, the GST-CTD_[52] sample turns turbid (middle). Microscopic images confirm the formation of liquid droplets (right images, top w/o dextran and bottom with dextran). The turbidity of 10 μM GST-CTD_[52] protein samples containing 9 or 52 hepta-repeats before and after addition of dextran was determined at 600 nm (OD₆₀₀). Scale bars, 25 μm. B, OD₆₀₀ measurements of phase-separated GST-CTD_[52] upon increasing concentrations of 1,6-hexanediol. Data represent mean ± SD from three replicates. C, dissolution of GST-CTD_[52] droplets by phosphorylation with Cdk7/CycH. In total, 10 μM GST-CTD_[52] was incubated with 0.1 μM Cdk7/CycH and 2 mM ATP in kinase assay buffer containing 16% dextran. The turbidity of the samples was determined at indicated time points in duplicate measurements. For analysis of the phosphorylation status, the reaction was quenched by mixing with 2× SDS sample buffer and subsequent analysis of 2 μg GST-CTD_[52] in a 12% SDS-PAGE. Microscopic images at three time points revealing the dissolution of condensates are shown at the bottom. Scale bars, 25 μm. D, at 5% 1,6-hexanediol GST-CTD_[52] droplets remain intact, whereas the kinase activity of Cdk7/CycH is impaired. The measurement was performed similarly as in C but in the presence of 5% 1,6-hexanediol. The stability of the condensates is confirmed by light microscopy images (bottom). Scale bars, 25 μm.