Interaction of elongation factors TFIIS and elongin A with a human RNA polymerase II holoenzyme capable of promoter-specific initiation and responsive to transcriptional activators.
Affinity chromatography on columns containing the immobilized monomeric transcriptional elongation factor TFIIS or the essential large subunit, Elongin A, of the trimeric elongation factor, Elongin, was used to purify a human RNA polymerase II holoenzyme from HeLa whole cell extract. This holoenzyme contained nearstoichiometric amounts of all the general transcription factors, TFIIB, TFIID (TBP + TAFIIs), TFIIE, TFIIF, and TFIIH, required to accurately initiate transcription in vitro at the adenovirus major late promoter. It behaved as a large complex, slightly smaller than 70 S ribosomes, during gel filtration chromatography, and contained nearly half the TFIID that was present in the extract used for the affinity chromatography. It also contained the cyclin-dependent kinase CDK8, a human homologue of the Saccharomyces cerevisiae holoenzyme subunit SRB10, and many other polypeptides. Efficient interaction of holoenzyme with TFIIS or Elongin A required only the amino-terminal region of either protein. These regions are similar in amino acid sequence but dispensable for TFIIS or Elongin to regulate elongation in vitro by highly purified RNA polymerase II. The transcriptional activators GAL4-VP16 and GAL4-Sp1 activated transcription in vitro by purified holoenzyme in the absence of any additional factors.
Pan
G
G
Banting and Best Department of Medical Research and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
Aso
T
T
Greenblatt
J
J
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D000075065:Elongin; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012319:RNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000494:pharmacology*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D035165:Transcription Factors, General; D035602:Transcriptional Elongation Factors
C000616397:ELOA protein, human; D000075065:Elongin; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D035165:Transcription Factors, General; D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II; D005982:Glutathione Transferase; D012319:RNA Polymerase II