FIG 4.

Effects of CTD mutations on transcription, RNAP II assembly, and subcellular distribution. (A) Cells were treated with Tet for 40 h, and nuclei were harvested. NRO assays were performed as described in the text. Slot blots contained the indicated DNA probes (top panel). NC1 and NC1 are two negative controls. Signals from each gene were normalized to 18S RNA and plotted (bottom panel) (n = 2). Error bars indicate standard deviations. (B) Rpb1 proteins were immunoprecipitated from cells cultured in the presence of Tet for 24 h. The content of Rpb2 in the RNAP II complex was determined by Western blotting. (C) Subcellular fractionation was performed in cells treated with Tet for 24 h. Rpb1 localization was determined by Western blotting. Nuclear protein U2AF65 and chromatin-bound histone H3 protein served as controls for fractionation.