Figure 4.

Tat increases the ability of CAK to phosphorylate the CTD of Pol II. (A) Wild-type HA-tagged CDK7 (CDK7), or a kinase-deficient mutant of CDK7 (M) were immunoprecipitated from stably expressing HeLa cells. GST–CTD protein (25 ng) was incubated with casein kinase (CK, 25 ng), or CDK7 immunoprecipitates in the presence of wild-type (Tat) or mutant (mTat = K41A) Tat proteins and γ-ATP. Kinase reaction products were subjected to SDS-PAGE, and gels were visualized by autoradiography after drying. Positions of the hypophosphorylated (CTD_a) and hyperphosphorylated (CTD_o) GST–CTD fusions, as well as cyclin H are indicated on the left. (B) GST–CTD was incubated with recombinant CAK (50 ng) in the presence of wild-type (Tat) or mutant (mTat = K41A) Tat proteins in a kinase reaction. Products were processed as described above. (C) Purified Pol II was incubated with CAK in the presence of wildtype (Tat) or mutant (mTat = K41A) Tat proteins in a kinase reaction. Products were processed as described above. (D) CyclinAΔ171/CDK2HA complexes bound to protein-A–Sepharose beads were incubated with CAK (50 ng) in the presence of wild-type (Tat) or mutant (mTat = K41A) Tat proteins. After washing the beads were incubated with histone H1 and kinase reactions allowed to proceed for 1 hr. Products were processed as described above. Position of labeled histone H1 (H1) is indicated.