FIG. 6.

The HAD domain is not required for CTD phosphorylation by the Ume3p–Ume5p kinase. (A) Ume5p phosphorylates the CTD in a Ume3p-dependent manner. Protein extracts prepared from strains with the indicated genotypes harboring either HA tagged Ume5p or Kin28p were immunoprecipitated using the HA mAb. Immunoprecipitates were incubated with CTD peptide and radiolabeled ATP. The phosphorylated CTD was visualized using SDS-PAGE and autoradiography (top panel). Aliquots of the immunoprecipitates were probed by Western blot analysis to verify the recovery of Ume5p or Kin28p (arrows, lower panel). (B) The HAD is dispensable for CTD phosphorylation. A ume3Δ strain harboring HA-tagged Ume5p and the indicated UME3 expression constructs were assayed for CTD kinase activity as described above. The levels of Ume5p and the Ume3p derivatives in the immunoprecipitates were determined by Western blot analysis.