Characterization of cDNA for the large subunit of the transcription initiation factor TFIIF

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Abstract
AT least six chromatographically resolvable general transcription factors may participate in accurate initiation by RNA polymerase II in HeLa cell-derived systems1–3. TFIIF (also termed FC, RAP30/74 and β/γ) can bind directly to RNA polymerase II in solution4–6 and decrease the affinity of RNA polymerase II for nonspecific DNA7. From studies on the kinetics of transcription initiation1, on the composition of transcription initiation complexes fractionated by acrylamide gel electrophoresis8, and on template competition experiments9, TFIIF is known to act at an intermediate stage in initiation complex formation. It acts after TFIID firmly associates with DNA, but coincidentally with or immediately after RNA polymerase II binding to DNA, and before the recruitment of factor TFIIE. TFIIF may6 or may not4,5 have DNA helicase activity. The small subunit (RAP30) of TFIIF has been cloned6 and shows some amino-acid sequence homology to bacterial cr factors. We have partially sequenced the RAP74 protein from purified HeLa cells, cloned its complementary DNA and shown that its translation product can interact with RAP30 in vitro as well as in vivo. The cDNA predicts an amino-acid sequence that lacks obvious DNA or RNA helicase motifs. It has regions rich in charged amino acids, including segments containing a higher content of acidic amino acids than are found in strong transcriptional activators such as VP16 (ref. 10).