Witte M M, Dickson R C
Department of Biochemistry, University of Kentucky, Lexington 40536-0084.
Mol Cell Biol. 1990 Oct;10(10):5128-37. doi: 10.1128/mcb.10.10.5128-5137.1990.
LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.
LAC9是一种DNA结合蛋白，可调节乳酸克鲁维酵母中乳糖 - 半乳糖操纵子的转录。DNA结合结构域由一个锌指和附近的氨基酸组成（M.M.威特和R.C.迪克森，《分子细胞生物学》8:3726 - 3733，1988年）。单个锌指在结构上似乎与许多其他真菌转录激活蛋白的锌指相关，这些蛋白含有带正电荷的残基和六个保守的半胱氨酸，其一般形式为Cys - Xaa2 - Cys - Xaa6 - Cys - Xaa6 - 9 - Cys - Xaa2 - Cys - Xaa 6 - Cys，其中Xaan表示一段指定数量的任意氨基酸（R.M.埃文斯和S.M.霍伦伯格，《细胞》52:1 - 3，1988年）。锌指和其他氨基酸在DNA结合中的功能仍不清楚。为了确定LAC9 DNA结合结构域的哪一部分介导序列识别，我们用酿酒酵母PPR1或LEU3蛋白的类似区域替换了C6锌指、锌指羧基侧相邻的氨基酸或两者。一种嵌合LAC9蛋白LAC9(PPR1 34 - 61)，只携带PPR1锌指，保留了LAC9的DNA结合特异性。然而，携带PPR1锌指和锌指羧基侧14个氨基酸的LAC9(PPR1 34 - 75)获得了PPR1的DNA结合特异性，表明这14个氨基酸对于特异性DNA结合是必需的。我们的数据表明，C6指可以相互替代并允许DNA结合，但结合亲和力降低。因此，从定性意义上讲，C6指执行类似的功能。然而，天然C6指蛋白所需的高亲和力结合需要具有特定氨基酸序列的独特C6指。这一要求可能反映了构象限制，包括C6指与羧基相邻氨基酸之间的相互作用；或者另外，这可能表明锌指中独特的、非保守的氨基酸残基与DNA形成了序列特异性或稳定的接触。
