FIGURE 4.

Loss of P-IIF from the PIC followed by challenge with Gdown1 results in inclusion of Gdown1 in the transcript elongation complex. PICs were prepared on the CMV template with 72 fmol of unphosphorylated (U) or CK2 phosphorylated (P) TFIIF per reaction as described under “Experimental Procedures.” A, PICs were low salt-rinsed (which removes only the P-IIF), incubated with a 20-fold excess of Gdown1 to pol II or with buffer, pulse-labeled, rinsed with high salt, and then chased with no addition, 166 fmol TFIIF, or P-IIF as described under “Experimental Procedures.” The schematic of the assay is shown below the panels. Note that the step of removing unbound Gdown1 from the PIC applies only to C. The white bar separates non-adjacent lanes within the same gel. The lengths of size markers are shown to the left of the panel. B, as in A, except that the PICs were not rinsed but used directly (in the assay schematic, the bracketed rinse step was removed). Lane X is the lower section of an adjacent lane, resulting from distortion in the gel. C, as described in A, except that the unbound Gdown1 in the supernatant (supernat.) was removed before the pulse. The schematic of the assay is shown below the panel. In this case, there was no low salt rinse step for the PIC.