Van Dyke M W, Sawadogo M
Department of Tumor Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030.
Mol Cell Biol. 1990 Jul;10(7):3415-20. doi: 10.1128/mcb.10.7.3415-3420.1990.
The existence of separable functions within the human class II general transcription factor TFIID was probed for differential sensitivity to mild proteolytic treatment. Independent of whether TFIID was bound to DNA or free in solution, partial digestion with either one of a variety of nonspecific endoproteases generated a protease-resistant protein product that retained specific DNA recognition, as revealed by DNase I footprinting. However, in contrast to native TFIID, which interacts with the adenovirus major late (ML) promoter over a very broad DNA region, partially proteolyzed TFIID interacted with only a small region of the ML promoter immediately surrounding the TATA sequence. This novel footprint was very similar to that observed with the TATA factor purified from yeast cells. Partially proteolyzed human TFIID could form stable complexes that were resistant to challenge by exogenous templates. It could also nucleate the assembly of transcription complexes on the ML promoter with an efficiency comparable to that of native TFIID, yielding similar levels of transcription initiation. These results suggest a model in which the human TFIID protein is composed of at least two different regions or polypeptides: a protease-resistant "core," which by itself is sufficient for promoter recognition and basal transcriptional levels, and a protease-sensitive "tail," which interacts with downstream promoter regions and may be involved in regulatory processes.
通过对温和蛋白酶处理的差异敏感性，探究了人类II类通用转录因子TFIID中可分离功能的存在情况。无论TFIID是与DNA结合还是游离于溶液中，用多种非特异性内切蛋白酶中的任何一种进行部分消化，都会产生一种抗蛋白酶的蛋白质产物，经DNase I足迹法显示，该产物保留了特异性DNA识别能力。然而，与在非常广泛的DNA区域与腺病毒主要晚期（ML）启动子相互作用的天然TFIID不同，部分蛋白酶解的TFIID仅与紧邻TATA序列的ML启动子的一小区域相互作用。这种新的足迹与从酵母细胞中纯化的TATA因子所观察到的非常相似。部分蛋白酶解的人类TFIID可以形成对外源模板挑战具有抗性的稳定复合物。它还能够以与天然TFIID相当的效率在ML启动子上启动转录复合物的组装，产生相似水平的转录起始。这些结果提示了一个模型，即人类TFIID蛋白至少由两个不同的区域或多肽组成：一个抗蛋白酶的“核心”，其自身足以实现启动子识别和基础转录水平；以及一个对蛋白酶敏感的“尾巴”，它与下游启动子区域相互作用，可能参与调控过程。
