Cho S, Wensink P C
Rosenstiel Center and the Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110, USA.
J Biol Chem. 1997 Feb 7;272(6):3185-9. doi: 10.1074/jbc.272.6.3185.
Drosophila yolk protein genes are regulated by doublesex male protein (DSXM) in males and doublesex female protein (DSXF) in females. Both proteins bind to the same DNA sites from which DSXM represses and DSXF activates transcription. The proteins are identical through 397 NH2-terminal amino acids that include domains for oligomerization and DNA binding. The remaining COOH termini are sex-specific and include an essential part of a second oligomerization domain. We report here mobility shift assays that examine the DNA binding properties of purified DSXM and DSXF. Dimers of DSXM and DSXF bind to a regulatory site, dsxA, with the same affinity (Kapp = 0.2 nM), specificity (specific/nonspecific approximately 1.2 x 10(4)), and dependence on monovalent and divalent cations. The DNA association rate constants also are indistinguishable (kon = 4.6 x 10(6) M-1 s-1) as are the several terms of the dissociation reaction. Dissociation has an intrinsic rate of koff = 5.1 x 10(-4) s-1 and other rate terms that depend on the free concentration of specific DNA binding sites (2.4 x 10(4) M-1 s-1) or nonspecific binding sites (2.4 M-1 s-1). This first order dependence on unbound DNA suggests that a direct transfer between DNAs is likely to occur when DSX proteins search for specific sites in the many short open DNA regions of chromatin. Overall, dimer binding to individual DNA sites appears to be determined by the sex-nonspecific part of the two proteins. We infer that the sex-specific oligomerization domains play roles in binding cooperativity to multiple DNA sites or in other protein:protein interactions.
果蝇卵黄蛋白基因在雄性中受双性雄性蛋白（DSXM）调控，在雌性中受双性雌性蛋白（DSXF）调控。这两种蛋白都结合到相同的DNA位点，DSXM在该位点抑制转录，而DSXF在该位点激活转录。这两种蛋白在397个氨基末端氨基酸上是相同的，这些氨基酸包含寡聚化和DNA结合结构域。其余的羧基末端是性别特异性的，并且包含第二个寡聚化结构域的重要部分。我们在此报告迁移率变动分析，该分析检测了纯化的DSXM和DSXF的DNA结合特性。DSXM和DSXF的二聚体以相同的亲和力（Kapp = 0.2 nM）、特异性（特异性/非特异性约为1.2×10⁴）以及对单价和二价阳离子的依赖性结合到调控位点dsxA。DNA结合速率常数也无法区分（kon = 4.6×10⁶ M⁻¹ s⁻¹），解离反应的几个项也是如此。解离的固有速率为koff = 5.1×10⁻⁴ s⁻¹，其他速率项取决于特定DNA结合位点的游离浓度（2.4×10⁴ M⁻¹ s⁻¹）或非特异性结合位点的游离浓度（2.4 M⁻¹ s⁻¹）。这种对未结合DNA的一级依赖性表明，当DSX蛋白在染色质的许多短开放DNA区域中搜索特定位点时，很可能在DNA之间发生直接转移。总体而言，二聚体与单个DNA位点的结合似乎由这两种蛋白的性别非特异性部分决定。我们推断，性别特异性寡聚化结构域在与多个DNA位点的结合协同作用或其他蛋白质 - 蛋白质相互作用中发挥作用。
