DETECTING PROTEIN-DNA INTERACTIONS INVIVO - DISTRIBUTION OF RNA-POLYMERASE ON SPECIFIC BACTERIAL GENES
DETECTING PROTEIN-DNA INTERACTIONS INVIVO - DISTRIBUTION OF RNA-POLYMERASE ON SPECIFIC BACTERIAL GENES
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DOI：
10.1073/pnas.81.14.4275
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发表时间：
1984-01-01
期刊：
影响因子：
--
通讯作者：
LIS, JT
中科院分区：
文献类型：
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作者：
GILMOUR, DS;LIS, JT
提出了一种用于确定蛋白质在染色体DNA的特定区段上的体内分布的方法。首先，通过用紫外线照射完整的细胞，蛋白质与DNA共价连接。其次，这些细胞在去污剂中被破坏，并且从裂解物中免疫沉淀出特定的蛋白质。第三，纯化与沉淀物中的蛋白质共价连接的DNA，并通过杂交进行测定。通过检查在大肠杆菌中RNA聚合酶与组成型表达的大肠杆菌的交联来测试该方法。cI基因，以及未诱导的和异丙基β- D-硫代半乳糖苷（IPTG）诱导的乳糖操纵子。正如预期的那样，免疫沉淀物中组成型表达基因的恢复依赖于细胞的照射和RNA聚合酶抗血清的加入。lac操纵子DNA的回收还需要在交联步骤之前用IPTG进行转录活化。还检查了沙门氏菌在野生型、衰减子突变体和启动子突变体菌株中的亮氨酸操纵子上的RNA聚合酶的分布。在体内的数据是在完全一致的预测衰减模型的监管。讨论了这些方法的分辨率、灵敏度和通用性。
An approach is presented for determining the in vivo distribution of a protein on specific segments of chromosomal DNA. First, proteins are joined covalently to DNA by irradiating intact cells with UV light. Second, these cells are disrupted in detergent, and a specific protein is immunoprecipitated from the lysate. Third, the DNA that is covalently attached to the protein in the precipitate is purified and assayed by hybridization. This approach was tested by examining the cross-linking in Escherichia coli of RNA polymerase to a constitutively expressed, .lambda. cI gene, and to the uninduced and isopropyl .beta.-D-thiogalactoside (IPTG)-induced lac operon. As expected, the recovery of the constitutively expressed gene in the immunoprecipitate is dependent on the irradiation of cells and on the addition of RNA polymerase antiserum. The recovery of the lac operon DNA also requires transcriptional activation with IPTG prior to the cross-linking step. The distribution of RNA polymerase on the leucine operon of Salmonella in wild-type, attenuator mutant and promoter mutant strains was also examined. The in vivo data are in complete agreement with the predictions of the attenuation model of regulation. The resolution, sensitivity and generality of these methods are discussed.