Omichinski J G, Trainor C, Evans T, Gronenborn A M, Clore G M, Felsenfeld G
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1676-80. doi: 10.1073/pnas.90.5.1676.
Sequence-specific DNA binding has been demonstrated for a synthetic peptide comprising only one of the two "finger"-like domains of the erythroid transcription factor GATA-1 (also termed Eryf-1, NF-E1, or GF-1). Quantitative analysis of gel-retardation assays yields a specific association constant of 1.2 x 10(8) M, compared with values of about 10(9) M for the full-length natural GATA-1 protein. By the use of peptides of various lengths, it was possible to delineate the smallest region necessary for specific binding. A single C-terminal finger of the double-finger motif is necessary but not sufficient for sequence-specific interaction. Basic amino acids located C-terminal to the finger (some more than 20 amino acids away) are also essential for tight binding. In addition to demonstrating that zinc is important for the formation of an active binding complex, we show that other ions, notably Fe2+, can fulfill this role. Our results make it clear that the GATA-1 metal binding motif is quite distinct from that found in the steroid hormone family and that GATA-1 is a member of a separate class of DNA binding proteins.
已证明，一种合成肽具有序列特异性DNA结合能力，该合成肽仅包含红系转录因子GATA-1（也称为Eryf-1、NF-E1或GF-1）两个“指状”结构域中的一个。凝胶阻滞分析的定量分析得出特异性结合常数为1.2×10⁸ M，而全长天然GATA-1蛋白的值约为10⁹ M。通过使用不同长度的肽，可以确定特异性结合所需的最小区域。双指基序的单个C端指对于序列特异性相互作用是必要的，但并不充分。位于该指C端的碱性氨基酸（有些距离超过20个氨基酸）对于紧密结合也至关重要。除了证明锌对于形成活性结合复合物很重要外，我们还表明其他离子，特别是Fe²⁺，也可以起到这一作用。我们的结果清楚地表明，GATA-1金属结合基序与类固醇激素家族中的基序截然不同，并且GATA-1是一类单独的DNA结合蛋白的成员。
