A SINGLE CDNA, HTFIIA/ALPHA, ENCODES BOTH THE P35 AND P19 SUBUNITS OF HUMAN TFIIA
A SINGLE CDNA, HTFIIA/ALPHA, ENCODES BOTH THE P35 AND P19 SUBUNITS OF HUMAN TFIIA
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DOI：
10.1101/gad.7.11.2220
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发表时间：
1993-11-01
影响因子：
10.5
通讯作者：
ROEDER, RG
中科院分区：
文献类型：
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作者：
DEJONG, J;ROEDER, RG
TFIIA是一种转录因子，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。通过使用迁移率变动分析，从HeLa细胞中纯化TFIIA作为35-、19-和12-kD亚基的复合物。使用寡核苷酸分离人cDNA克隆hTFIIA/α，其编码与酵母基因TOA 1的产物具有同源性的55-kD蛋白。hTFIIA/α的开放阅读框含有从天然人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，抗血清提出的55-kD hTFIIA/α编码的蛋白质与p35和p19亚基的天然TFIIA反应，和重组蛋白可以在功能上取代这些亚基的迁移率变化测定与复性p12。通过hTFIIA/α蛋白的序列表明了对天然人TFIIA的有效亲和纯化，并在生物化学上证明了这一点。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，并通过再加入纯化的人TFIIA恢复到原始水平。
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.