Maxon M E, Tjian R
Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9529-33. doi: 10.1073/pnas.91.20.9529.
The functions of individual basal transcription factors during the formation of an initiation complex by RNA polymerase II remain largely unknown. Transcription factor IIE (TFIIE) has recently been shown to bind to multiple targets in the initiation complex. To assess the role of zinc binding in basal transcription, we have mutated the predicted zinc-finger domain of human TFIIE. Atomic absorption spectroscopy using purified recombinant proteins revealed that the large subunit, TFIIE-56, is indeed a zinc-binding protein. Mutation of a cysteine residue in the putative zinc-finger domain abolished zinc binding. Moreover, mutant TFIIE-56 failed to support reconstituted basal transcription in vitro, suggesting that zinc binding is required for TFIIE function. However, gel-filtration experiments and protein affinity experiments suggest that mutant TFIIE-56 forms a stable heterotetramer with the small subunit, TFIIE-34, that is similar to wild type. Interestingly, gel mobility shift experiments reveal that loss of transcriptional activity by mutant TFIIE is correlated with its inability to stably assemble into the transcription complex. These findings establish that zinc binding by TFIIE may help form a specific structure that is required for stable entry into the transcription complex.
在RNA聚合酶II形成起始复合物的过程中，各个基础转录因子的功能在很大程度上仍不清楚。转录因子IIE（TFIIE）最近被证明能与起始复合物中的多个靶点结合。为了评估锌结合在基础转录中的作用，我们对人TFIIE的预测锌指结构域进行了突变。使用纯化的重组蛋白进行的原子吸收光谱分析表明，大亚基TFIIE-56确实是一种锌结合蛋白。假定锌指结构域中的一个半胱氨酸残基发生突变会消除锌结合。此外，突变型TFIIE-56在体外无法支持重组基础转录，这表明锌结合是TFIIE功能所必需的。然而，凝胶过滤实验和蛋白质亲和力实验表明，突变型TFIIE-56与小亚基TFIIE-34形成了一种稳定的异源四聚体，与野生型相似。有趣的是，凝胶迁移率变动实验表明，突变型TFIIE转录活性的丧失与其无法稳定组装到转录复合物中有关。这些发现表明，TFIIE与锌的结合可能有助于形成一种特定结构，这是稳定进入转录复合物所必需的。
