A SENSITIVE METHOD FOR THE DETERMINATION OF PROTEIN-DNA BINDING SPECIFICITIES
A SENSITIVE METHOD FOR THE DETERMINATION OF PROTEIN-DNA BINDING SPECIFICITIES
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DOI：
10.1093/nar/18.21.6197
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发表时间：
1990-11-11
影响因子：
14.9
通讯作者：
TREISMAN, R
中科院分区：
文献类型：
--
作者：
POLLOCK, R;TREISMAN, R
我们描述了一种灵敏而快速的方法来确定DNA结合蛋白的序列特异性。该方法允许利用由无细胞翻译反应产生的粗细胞提取液中存在的少量蛋白质来恢复特定的位点。提取的蛋白质与随机序列寡核苷酸、免疫沉淀纯化的复合体以及通过聚合酶链式反应(PCR)扩增的结合DNA一起孵育。然后，这种DNA被用于进一步的结合、免疫沉淀和扩增，直到可以检测到特定的结合。以转录因子SRF为模型系统，我们证明了真实的高亲和力结合位点被恢复，并表明当特定抗体不可用时，可以使用表位标记来恢复位点。我们还表明，Fos蛋白结合的特定位点很容易通过这种技术恢复，Fos蛋白只有在与其他多肽复合时才与DNA高亲和力结合。
We describe a sensitive and rapid method for determination of the sequence specificity of DNA binding proteins. The method allows recovery of specific sites using the small amounts of protein present in crude cell extracts of produced by cell-free translation reactions. Extract proteins are incubated with a pool of random sequence oligonucleotides, complexes purified by immunoprecipitation, and bound DNA amplified by the Polymerase Chain Reaction (PCR). This DNA is then used in further rounds of binding, immunoprecipitation, and amplificaton, until specific binding is detectable. With the transcription factor SRF as a model system, we demonstrate that authentic high affinity binding sites are recovered, and show that epitope tagging can be used to allow recovery of sites when specific antibodies are unavailable. We also show that specific sites bound by the Fos protein, which binds DNA with high affinity only when complexed with other polypeptides, are easily recovered by this technique.