SEQUENCE-SPECIFIC RECOGNITION OF DNA BY ZINC-FINGER PEPTIDES DERIVED FROM THE TRANSCRIPTION FACTOR SP1
SEQUENCE-SPECIFIC RECOGNITION OF DNA BY ZINC-FINGER PEPTIDES DERIVED FROM THE TRANSCRIPTION FACTOR SP1
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DOI：
10.1073/pnas.89.20.9759
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发表时间：
1992-10-15
影响因子：
11.1
通讯作者：
CARADONNA, JP
中科院分区：
文献类型：
--
作者：
KRIWACKI, RW;SCHULTZ, SC;CARADONNA, JP
我们已经过度表达和纯化了Sp1的两个肽片段，其中包含特异性Sp1 DNA结合所必需的三个“锌指”结构域。DNA结合实验和核磁共振光谱显示，这些肽在溶液中具有稳定的折叠构象，存在Zn2+。迁移转移实验表明Sp1肽识别许多不同的Sp1 DNA结合位点（GC盒，核心序列为ggggcgg）。与GGGGCGGGGC序列结合的92个氨基酸的肽解离常数（K(d)几乎等于10 nM）以及与其他几个DNA序列的相对亲和力明确地显示了sp1样结合特性。Sp1-Zn92的热力学结合位点通过引物延伸/迁移/移位测定得到，GC盒DNA序列的5‘部分（GGG GCG）比3’部分（GGGC）对总结合能的贡献更大。这些发现是在Sp1氨基酸序列的背景下解释的，并与结构表征的Zif-268/DNA复合物进行了比较。本文提出了一个模型，该模型提供了Sp1识别不同DNA序列的能力的结构解释，根据三个锌指结构域的每个个体（和不同）DNA结合特性。
We have overexpressed and purified two peptide fragments of Sp1 that contain the three "zinc-finger" domains necessary for specific Sp1 DNA binding. These peptides assume a stable, folded conformation in solution in the presence of Zn2+ as shown by DNA binding assays and NMR spectroscopy. Mobility-shift assays demonstrate that the Sp1 peptides recognize a number of different Sp1 DNA binding sites (GC boxes, with the core sequence GGGCGG). The dissociation constant for a 92-amino acid peptide binding to the GGGGCGGGGC sequence (K(d) almost-equal-to 10 nM) and the relative affinities for several other DNA sequences definitively demonstrate Sp1-like binding properties. The thermodynamic binding site for Sp1-Zn92 has been mapped using the primer-extension/mobility/shift assay revealing that the 5' portion of the GC box DNA sequence (GGG GCG) contributes more strongly to the total binding energy than the 3' portion (GGGC). These findings are interpreted in the context of the Sp1 amino acid sequence in comparison with the structurally characterized Zif-268/DNA complex. A model is proposed that offers a structural explanation for the ability of Sp1 to recognize a diverse array of DNA sequences in terms of the individual (and different) DNA binding properties of each of the three zinc-finger domains.