Fig. 3.

Fcp1 induces loss of heparin binding by Pol II in a Mg²⁺-dependent manner. (A) Heparin-binding behavior of S. cerevisiae 12-subunit (wtPol II) in the absence and presence of S. cerevisiae Fcp1 (scFcp1). Consistent amounts of the polymerase were loaded onto heparin Sepharose columns with or without the Fcp1, indicated respectively on the top. Consistent volumes of the flow-through (F.T.) and high-salt elution (Elut) were collected. Contents of the two fractions were analyzed by SDS/PAGE with polypeptides (marked to the right) revealed by Coomassie staining. (B) (Upper) Heparin-binding behaviors of S. cerevisiae 12-subunit and 10-subunit (Δ4/7) Pol II were characterized by measuring their amounts that flowed through the microcolumns as functions of the presence of S. pombe Fcp1 (spFcp1) and BSA. The experiments were performed in the presence of Mg²⁺ (5–10 mM). (Lower) Proteins bound to the column after washing were eluted with high salt. The SDS gels were all stained with Coomassie blue and quantified. Relative molar amounts of the loading materials were indicated on the top. Protein bands are identified on the right. Presence of a polymerase in a fraction is revealed by its two largest subunits (Rpb1 and Rpb2). Note the contaminating bands from the BSA sample do not match with the positions of Rpb1 and Rpb2, although they are of high molecular mass. (C) Loss of heparin binding by Pol IIB in the presence of S. Pombe Fcp1 measured with the same assay. “Rpb1b” corresponds to the largest, CTD-less subunit of S. cerevisiae Pol II. WT Pol II (wtPol II) was loaded as a marker in lane 5. (D) Quantification (in ratios) of the gels in B and C. The amount of the polymerase relative to the control (lane 1 of B) in the flow-through was a function of the Fcp1/polymerase molar ratio. For four independent experiments, intensities of Rpb1 bands in lanes 5–8 of B were normalized relative to that of WT Pol II alone in lane 1, which was low but not zero. The control for Δ4/7 alone was not included in the assay but was assumed to be identical to the WT Pol II control. Quantification of Rpb1b in the flow-through of C was done in the same manner, except that it was based on a single experiment. Labels on the top of the bars indicate the corresponding gel lanes in B or C.(E) The effect of Mg²⁺ ion on heparin binding by Pol II. The assay was run with an Fcp1/Pol molar ratio of 1.0. In the absence of MgCl₂, the relative amount of Pol II that flowed through the heparin column dramatically reduced as judged by Coomassie staining (compare lanes 1 and 2).