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首页> 外文期刊>Biochemistry >Site-specific inhibition of transcription factor binding to DNA by a metallointercalator.
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Site-specific inhibition of transcription factor binding to DNA by a metallointercalator.

机译：金属嵌入剂对转录因子结合DNA的位点特异性抑制。

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摘要

The metallointercalator Lambda-1-Rh(MGP)2phi5+ binds tightly and specifically to the site 5'-CATATG-3' in the major groove of double helical DNA by a combination of direct readout and shape selection. To examine competitive interactions between this small metal complex and a DNA-binding transcription factor, the preferred binding site for Lambda-1-Rh(MGP)2phi5+ was engineered into the AP-1 recognition element (ARE) of the major-groove binding bZIP transcription factor yAP-1, the yeast analogue of mammalian AP-1. Binding experiments confirmed that the modified ARE retained normal yAP-1 binding affinity. Photocleavage experiments demonstrated that the modified ARE contained a high-affinity binding site for Lambda-1-Rh(MGP)2phi5+, whereas the native ARE showed no interaction. Competition experiments using gel shift mobility assays demonstrated that Lambda-1-Rh(MGP)2phi5+ at 120 nM competes 50% of yAP-1 binding to the 5'-CATATG-3' containing oligonucleotide. In contrast, competitive disruption of protein binding to the native ARE requires 3 microM Lambda-1-Rh(MGP)2phi5+. Metallointercalator derivatives, including geometric isomers of Lambda-1-Rh(MGP)2phi5+, show no specific binding to the target site and show no inhibition of yAP-1/DNA complexes at concentrations as high as 20 microM. Thus, metallointercalators can be tuned to show selectivity for major groove sites on DNA comparable to transcription factors and indeed can inhibit transcription factor binding site selectively.
机译：金属嵌入剂Lambda-1-Rh（MGP）2phi5 +通过直接读出和形状选择的组合紧密地且特异性地结合到双螺旋DNA主槽中的5'-CATATG-3'位。为了检查这种小金属配合物与DNA结合转录因子之间的竞争性相互作用，将Lambda-1-Rh（MGP）2phi5 +的首选结合位点工程化为主要凹槽结合bZIP的AP-1识别元件（ARE）转录因子yAP-1，哺乳动物AP-1的酵母类似物。结合实验证实，修饰的ARE保留了正常的yAP-1结合亲和力。光裂解实验表明，修饰的ARE包含Lambda-1-Rh（MGP）2phi5 +的高亲和力结合位点，而天然ARE没有相互作用。使用凝胶迁移迁移率测定法的竞争实验表明，在120 nM处的Lambda-1-Rh（MGP）2phi5 +与yAP-1结合50％的含5'-CATATG-3'的寡核苷酸竞争。相反，竞争性破坏蛋白质与天然ARE的结合需要3 microM Lambda-1-Rh（MGP）2phi5 +。金属嵌入剂衍生物，包括Lambda-1-Rh（MGP）2phi5 +的几何异构体，在高至20 microM的浓度下未显示与靶位点的特异性结合，也未显示对yAP-1 / DNA复合物的抑制作用。因此，可以调节金属嵌入剂以显示对DNA上与转录因子相当的主要凹槽位点的选择性，并且确实可以选择性抑制转录因子结合位点。

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