Identifying a transcription factor interaction site on RNA polymerase II

We have generated a series of fusion proteins carrying portions of subunit IIc, the second largest subunit of Drosophila RNA polymerase I, and have used them in a domain interference assay to identify a fragment of the IIc subunit that carries the binding site for a basal transcription factor. Fusion proteins carrying a subunit IIc fragment spanning residues Ala519-Gly992 strongly inhibit promoter-driven transcription in both unfractionated nuclear extracts and in reconstituted systems. The same fusion proteins similarly inhibit dTFIIF stimulation of Pol II elongation on dC-tailed templates, suggesting that the IIc(A519-G992) fragment, which carries conserved regions D-H, interferes with transcription by binding to dTFIIF. Finally, dTFIIF can be specifically cross-linked to a GST-IIc(A519-G992) fusion protein or to subunit IIc in intact Pol II.