DeJong J, Roeder R G
Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York 10021.
Genes Dev. 1993 Nov;7(11):2220-34. doi: 10.1101/gad.7.11.2220.
TFIIA is a transcription factor that, by interacting with the TATA-binding subunit (TBP) of TFIID, modulates transcription initiation by RNA polymerase II in vitro. By use of a mobility shift assay, TFIIA was purified from HeLa cells as a complex of 35-, 19-, and 12-kD subunits. Oligonucleotides were used to isolate a human cDNA clone, hTFIIA/alpha, which encodes a 55-kD protein with homology to the product of the yeast gene TOA1. The open reading frame of hTFIIA/alpha contains peptide sequences obtained from both the p35 and p19 subunits of natural human TFIIA, and thus encodes these two subunits. Consistent with this, antiserum raised against the 55-kD hTFIIA/alpha-encoded protein reacted with both the p35 and p19 subunits of natural TFIIA, and the recombinant protein could functionally replace those subunits in a mobility shift assay with renatured p12. An efficient affinity purification for natural human TFIIA was suggested by the sequence of the hTFIIA/alpha protein and demonstrated biochemically. Finally, transcription from the adenovirus major late promoter was greatly reduced in nuclear extracts depleted with anti-TFIIA/alpha serum and was restored to original levels by the readdition of purified human TFIIA.
TFIIA是一种转录因子，它通过与TFIID的TATA结合亚基（TBP）相互作用，在体外调节RNA聚合酶II的转录起始。利用迁移率变动分析，从HeLa细胞中纯化出TFIIA，它是由35kD、19kD和12kD亚基组成的复合物。使用寡核苷酸分离出一个人cDNA克隆hTFIIA/α，它编码一种与酵母基因TOA1产物具有同源性的55kD蛋白质。hTFIIA/α的开放阅读框包含从天然人TFIIA的p35和p19亚基获得的肽序列，因此编码这两个亚基。与此一致的是，针对55kD hTFIIA/α编码蛋白产生的抗血清与天然TFIIA的p35和p19亚基都发生反应，并且在与复性的p12进行的迁移率变动分析中，重组蛋白可以在功能上替代那些亚基。hTFIIA/α蛋白的序列提示并通过生化方法证明了对天然人TFIIA的有效亲和纯化。最后，在用抗TFIIA/α血清耗尽的核提取物中，腺病毒主要晚期启动子的转录大大降低，通过重新添加纯化的人TFIIA可恢复到原始水平。
