Culotta V C, Hsu T, Hu S, Fürst P, Hamer D
Laboratory of Biochemistry, National Cancer Institute, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1989 Nov;86(21):8377-81. doi: 10.1073/pnas.86.21.8377.
We describe a cell-free system in which the transcription of the yeast metallothionein gene is inducible by the addition of metal ions plus a specific regulatory protein. Efficient transcription requires the complete yeast ACE1 metalloregulatory protein, including both its DNA-binding and transactivation domains; a mouse nuclear extract providing RNA polymerase and general transcription factors; a template containing the ACE1 binding site; and Cu(I). Because the binding of ACE1 to DNA is dependent on Cu, it is possible to inhibit transcription by the use of Cu-complexing agents such as CN-. We have used this specific inhibition to show that the ACE1 regulatory protein is required for the maintenance as well as the formation of a functional preinitiation complex. The ability to reversibly induce yeast metallothionein gene transcription in vitro provides a powerful system for determining the molecular mechanism of a simple eukaryotic regulatory circuit.
我们描述了一种无细胞系统，在该系统中，通过添加金属离子和一种特定的调节蛋白可诱导酵母金属硫蛋白基因的转录。高效转录需要完整的酵母ACE1金属调节蛋白，包括其DNA结合结构域和反式激活结构域；提供RNA聚合酶和通用转录因子的小鼠核提取物；包含ACE1结合位点的模板；以及Cu(I)。由于ACE1与DNA的结合依赖于Cu，因此可以通过使用诸如CN-等Cu络合剂来抑制转录。我们利用这种特异性抑制作用表明，ACE1调节蛋白对于功能性起始前复合物的维持以及形成都是必需的。体外可逆诱导酵母金属硫蛋白基因转录的能力为确定简单真核调节回路的分子机制提供了一个强大的系统。
