Charge neutralization and DNA bending by the Escherichia coli catabolite activator protein
Charge neutralization and DNA bending by the Escherichia coli catabolite activator protein
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DOI：
10.1093/nar/30.9.1879
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发表时间：
2002-05-01
影响因子：
14.9
通讯作者：
Maher, LJ
中科院分区：
文献类型：
--
作者：
Hardwidge, PR;Zimmerman, JM;Maher, LJ
我们感兴趣的是不对称磷酸中和蛋白质诱导的DNA弯曲中的作用。我们描述了一个实验估计的实际静电贡献的不对称磷酸中和弯曲的DNA由大肠杆菌分解代谢物激活蛋白（CAP），一个原型的DNA弯曲蛋白。根据X-射线晶体结构，CAP和DNA磷酸盐之间假定的静电相互作用分配后，适当的磷酸盐在CAP半位点DNA被甲基膦酸酯取代化学中和。然后使用半合成DNA电泳定相测定评价DNA形状。我们的结果证实了未修饰的CAP DNA半位点序列在与蛋白质复合物中的增强方向上固有地弯曲了26度。在缺乏蛋白质的情况下，中和五种适当的磷酸盐可以使DNA的弯曲度增加到32度（类似于增加23%），与预测的方向一致。改变中和磷酸盐的位置会改变DNA的形状，这表明序列导向的DNA曲率可以通过磷酸盐中和的不对称性来改变。我们认为，不对称磷酸中和有助于DNA弯曲的CAP，但不能占完整的DNA变形。
We are interested in the role of asymmetric phosphate neutralization in DNA bending induced by proteins. We describe an experimental estimate of the actual electrostatic contribution of asymmetric phosphate neutralization to the bending of DNA by the Escherichia coli catabolite activator protein (CAP), a prototypical DNA-bending protein. Following assignment of putative electrostatic interactions between CAP and DNA phosphates based on X-ray crystal structures, appropriate phosphates in the CAP half-site DNA were chemically neutralized by methylphosphonate substitution. DNA shape was then evaluated using a semi-synthetic DNA electrophoretic phasing assay. Our results confirm that the unmodified CAP DNA half-site sequence is intrinsically curved by 26degrees in the direction enhanced in the complex with protein. In the absence of protein, neutralization of five appropriate phosphates increases DNA curvature to 32degrees (similar to23% increase), in the predicted direction. Shifting the placement of the neutralized phosphates changes the DNA shape, suggesting that sequence-directed DNA curvature can be modified by the asymmetry of phosphate neutralization. We suggest that asymmetric phosphate neutralization contributes favorably to DNA bending by CAP, but cannot account for the full DNA deformation.