DNA binding by the male and female doublesex proteins of Drosophila melanogaster
DNA binding by the male and female doublesex proteins of Drosophila melanogaster
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DOI：
10.1074/jbc.272.6.3185
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发表时间：
1997-02-07
影响因子：
4.8
通讯作者：
Wensink, PC
中科院分区：
文献类型：
--
作者：
Cho, S;Wensink, PC
果蝇卵黄蛋白基因受雄性双性蛋白(Dsx(M))和雌性双性蛋白(Dsx(F))的调控。这两种蛋白质都与DsX(M)抑制和DsX(F)激活转录的DNA位点结合，这两种蛋白质通过397个NH2末端氨基酸相同，包括寡聚和DNA结合结构域。其余的COOH末端是性别特异的，包括第二个低聚结构域的重要部分，我们报告了迁移率改变分析，检测了纯化的dsx(M)和dsx(F)，dsx(M)和dsx(F)的二聚体与调节位点dsxA的结合性质，具有相同的亲和力(K-app=0.2 nm)、特异性(特异性/非特异性约为1.2×10(4))以及对单价和二价阳离子的依赖性。与解离反应的几个项一样，缔合速率常数也是不可区分的(k(On)=4.6x10(6)M(-1)S(-1))。解离的本征速率k(Off)=5.1x10(-4)S(-1)，其他速率项取决于特定DNA结合位点(2.4x10(-4)M(-1)S(-1))或非特异结合位点(2.4M(-1)S(-1))的自由浓度。这种对非结合DNA的一级依赖性表明，当dsx蛋白在染色质的许多短的开放DNA区域中寻找特定的位置时，DNA之间的直接转移很可能发生。总体而言，二聚体与单个DNA位点的结合似乎是由这两种蛋白质的性别非特异性部分决定的，我们推测，性别特异的寡聚化结构域在与多个DNA位点或其他蛋白质：蛋白质相互作用的结合协同性中发挥作用。
Drosophila yolk protein genes are regulated by doublesex male protein (DSX(M)) in males and doublesex female protein (DSX(F)) in females. Both proteins bind to the same DNA sites from which DSX(M) represses and DSX(F) 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 DSX(M) and DSX(F), Dimers of DSX(M) and DSX(F) bind to a regulatory site, dsxA, with the same affinity (K-app = 0.2 nM), specificity (specific/nonspecific approximate to 1.2 x 10(4)), and dependence on monovalent and divalent cations. The DNA association rate constants also are indistinguishable (k(on) = 4.6 x 10(6) M(-1) s(-1)) as are the several terms of the dissociation reaction. Dissociation has an intrinsic rate of k(off) = 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.