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1 Graduate School of Frontier Biosciences, and
2 Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan
3 Laboratory of Gene Regulation, Graduate School of Medical and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194, Japan
4 Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10021, USA
The human thyroid hormone receptor-associated proteins (TRAP)/Mediator and related complexes mediate transcription through regulatory factors. To further understand the structural and functional diversity of these complexes we established three HeLa cell lines each expressing one of three epitope-tagged human TRAP/Mediator subunits, MED6, MED7, and CDK8 and isolated the complexes in which these subunits were contained by affinity and HPLC-gel filtration chromatography. The largest complexes from each cell line had a molecular mass of 1.5 MDa and possessed almost identical subunit compositions; we designated these complexes TRAP/Mediator-like complex 1 (TMLC1). Two potential subcomplexes were additionally observed: a 1-MDa complex from the CDK8-cell line (TMLC2) and a 600-kDa complex from the MED6-cell line (TMLC3). All three complexes regulated transcription in vitro; TMLC1 and TMLC3 augmented transcriptional activation, whereas TMLC2 repressed it. TMLC1 and TMLC2 phosphorylated RNA polymerase II (Pol II), but TMLC3 did not. Furthermore, TMLC1 predominantly interacted with the general transcription factors TFIIE, TFIIF, and TFIIH, which function during transcription initiation and the transition to elongation. In a final experiment, knockdown of CDK8 using RNA interference prevented transcriptional activation by Gal4-VP16 in a luciferase-assay. This, together with the effect of TMLC1 on transcription in vitro, suggests that CDK8 play positive roles in transcriptional activation.
aPresent Address: Department of Medical Technology, Faculty of Health Sciences, Kobe University School of Medicine, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142, Japan * Correspondence: E-mail: ohkumay{at}pha.u-toyama.ac.jp
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