INTERACTION OF ELONGATION-FACTORS TFIIS AND ELONGIN-A WITH A HUMAN RNA-POLYMERASE-II HOLOENZYME CAPABLE OF PROMOTER-SPECIFIC INITIATION ANDRESPONSIVE TO TRANSCRIPTIONAL ACTIVATORS

Affinity chromatography on columns containing the immobilized monomeri c transcriptional elongation factor TFIIS or the essential large subun it, Elongin A, of the trimeric elongation factor, Elongin, was used to purify a human RNA polymerase II holoenzyme from HeLa whole cell extr act. This holoenzyme contained near-stoichiometric amounts of all the general transcription factors, TFIIB, TFIID (TBP + TAF(II)s), TFIIE, T FIIF, and TFIIH, required to accurately initiate transcription in vitr o at the adenovirus major late promoter. It behaved as a large complex , slightly smaller than 70 S ribosomes, during gelfiltration chromatog raphy, and contained nearly half the, TFIID that was present in the ex tract used for the affinity chromatography. It also contained the cycl in-dependent kinase CDK8, a human homologue of the Saccharomyces cerev isiae holoenzyme subunit SRB10, and many other polypeptides. Efficient interaction of holoenzyme with TFIIS or Elongin A required only the a mino-terminal region of either protein. These regions are similar in a mino acid sequence but dispensable for TFIIS or Elongin to regulate el ongation in, vitro by highly purified RNA polymerase II. The transcrip tional activators GAL4-VP16 and GAL4-Sp1 activated transcription in vi tro by purified holoenzyme in the absence of any additional factors.