Pan G, Aso T, Greenblatt J
Banting and Best Department of Medical Research and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
J Biol Chem. 1997 Sep 26;272(39):24563-71. doi: 10.1074/jbc.272.39.24563.
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.
利用固定化单体转录延伸因子TFIIS或三聚体延伸因子Elongin的必需大亚基Elongin A的柱亲和层析，从HeLa全细胞提取物中纯化人RNA聚合酶II全酶。这种全酶含有接近化学计量的所有一般转录因子，即TFIIB、TFIID（TBP + TAFIIs）、TFIIE、TFIIF和TFIIH，这些因子是在体外腺病毒主要晚期启动子处准确起始转录所必需的。在凝胶过滤层析过程中，它表现为一种大复合物，略小于70 S核糖体，并且含有用于亲和层析的提取物中近一半的TFIID。它还含有细胞周期蛋白依赖性激酶CDK8，即酿酒酵母全酶亚基SRB10的人同源物，以及许多其他多肽。全酶与TFIIS或Elongin A的有效相互作用仅需要这两种蛋白质的氨基末端区域。这些区域的氨基酸序列相似，但对于TFIIS或Elongin通过高度纯化的RNA聚合酶II在体外调节延伸来说并非必需。转录激活因子GAL4-VP16和GAL4-Sp1在没有任何其他因子的情况下，通过纯化的全酶在体外激活转录。
