FIG. 6.

Comparison of P-TEFb and CAK phosphorylation of SPT5 constructs. (A) Purified RNA polymerase II (Pol II) was assayed in in vitro kinase assays using RNA polymerase II alone (lane 1) or in the presence of baculovirus-produced and purified CAK including CDK7, cyclin H, and MAT1 (lane 2) or CDK9 and cyclin T1 (lane 3). MW, molecular weight in thousands. (B) In vitro kinase assays were performed using baculovirus-produced preparations of CDK9/cyclin T1 (lanes 1 to 6) or CAK (lanes 7 to 12) with either no added substrate (lanes 1 and 7), GST-SPT5 (lanes 2 and 8), GST-SPT5 (aa 1 to 754) (lanes 3 and 9), GST-SPT5 (aa 760 to 852) (lanes 4 and 10), GST-SPT5 (aa 814 to 1087) (lanes 5 and 11), or GST-SPT5 (aa 760 to 1087) (lanes 6 and 12). Phosphorylation was assayed following SDS-PAGE and autoradiography. (C) A Coomassie blue-stained gel of the various GST-SPT5 fusion proteins used in the in vitro kinase assays in part B is shown.