Importance of a flanking AT-rich region in target site recognition by the GC box-binding zinc finger protein MIG1
Importance of a flanking AT-rich region in target site recognition by the GC box-binding zinc finger protein MIG1
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DOI：
10.1128/mcb.14.3.1979-1985.1994
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发表时间：
1994-03
影响因子：
5.3
通讯作者：
M. Lundin;J. Nehlin;H. Ronne
中科院分区：
文献类型：
--
作者：
M. Lundin;J. Nehlin;H. Ronne
MIG 1是一种锌指蛋白，在酿酒酵母中介导葡萄糖阻遏。MIG 1与哺乳动物的Krox/Egr、Wilms肿瘤和Sp1指蛋白相关。它有两个指，并结合到GCGGGG基序，类似于这些哺乳动物蛋白识别的GC盒。我们已经进行了一个完整的饱和诱变的天然MIG 1位点，以阐明其结合特异性。我们发现GC盒中只有三个突变保留了结合MIG 1的能力：G1与C，C2与T，G5与A。这一结果与目前的锌指-DNA结合模型一致，该模型假设序列特异性由GC盒内的碱基三联体识别决定。令人惊讶的是，我们发现GC盒5'端富含AT的区域对MIG 1结合也很重要。该AT盒存在于所有天然的MIG 1位点中，并且在DNase I足迹中受到MIG 1的保护。然而，AT盒与GC盒的不同之处在于，AT盒中没有单个碱基对于结合是必需的。相反，这个序列富含AT的性质似乎是至关重要的。已知富含AT的序列可以增加DNA的灵活性，这一事实促使我们测试MIG 1是否会弯曲DNA。我们发现，结合的MIG 1与弯曲的AT盒。我们的结论是，DNA结合一个简单的锌指蛋白，如MIG 1可以涉及识别的GC盒和侧翼序列的偏好，可能反映当地的DNA弯曲。
MIG1 is a zinc finger protein that mediates glucose repression in the yeast Saccharomyces cerevisiae. MIG1 is related to the mammalian Krox/Egr, Wilms' tumor, and Sp1 finger proteins. It has two fingers and binds to a GCGGGG motif that resembles the GC boxes recognized by these mammalian proteins. We have performed a complete saturation mutagenesis of a natural MIG1 site in order to elucidate its binding specificity. We found that only three mutations within the GC box retain the ability to bind MIG1: G1 to C, C2 to T, and G5 to A. This result is consistent with current models for zinc finger-DNA binding, which assume that the sequence specificity is determined by base triplet recognition within the GC box. Surprisingly, we found that an AT-rich region 5' to the GC box also is important for MIG1 binding. This AT box is present in all natural MIG1 sites, and it is protected by MIG1 in DNase I footprints. However, the AT box differs from the GC box in that no single base within it is essential for binding. Instead, the AT-rich nature of this sequence seems to be crucial. The fact that AT-rich sequences are known to increase DNA flexibility prompted us to test whether MIG1 bends DNA. We found that binding of MIG1 is associated with bending within the AT box. We conclude that DNA binding by a simple zinc finger protein such as MIG1 can involve both recognition of the GC box and flanking sequence preferences that may reflect local DNA bendability.