Figure 4.

CTD kinase activities of isolated Drosophila dCDK12 or human hCDK13 enzymes. (A) dCDK12 was eluted from antibody beads using the antigenic peptide, and elution fractions (E1, E2, and E3) were assayed for kinase activity using a βgal-dCTD fusion protein as substrate (apparent molecular weight ∼145 kDa). Coomassie-stained gel of terminated reactions is shown on the left, aligned with the corresponding autoradiogram on the right. (Lanes E1–E3) dCDK12 elutions all show CTD kinase (hyperphosphorylation) activity, as evidenced by the signals in the autoradiogram. No signal is observed in the no-enzyme control lane (fourth lane, no E). (B) dCDK12 was purified using protein A/G beads saturated with affinity-purified anti-dCDK12 IgG. After extensive washing, including a 0.8 M NaCl wash (see the Materials and Methods), the beads were assayed for CTD kinase activity using a GST-yCTD fusion protein as substrate. The Coomassie-stained gel on the left is aligned with the autoradiogram on the right. CTD kinase activity is observed (mobility-shifted band above GST-yCTD position) only when both beads (dCDK12) and CTD substrate are present. (C) hCDK13 was isolated using ion-exchange chromatography followed by immunoaffinity purification (Materials and Methods); the antibody beads were washed extensively and assayed as in B. The mobility-shifted band (autoradiogram) above GST-yCTD position indicates hyperphosphorylation of CTD.