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.
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004330:Drosophila / Q000235:genetics; D017344:Genes, Insect; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D012319:RNA Polymerase II; D001616:beta-Galactosidase