Interaction of the bovine papillomavirus type 1 E2 transcriptional control protein with the viral enhancer: purification of the DNA-binding domain and analysis of its contact points with DNA
Interaction of the bovine papillomavirus type 1 E2 transcriptional control protein with the viral enhancer: purification of the DNA-binding domain and analysis of its contact points with DNA
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牛乳头瘤病毒 1 型 E2 转录控制蛋白与病毒增强子的相互作用：DNA 结合域的纯化及其与 DNA 的接触点分析
DOI：
10.1128/jvi.62.6.1925-1931.1988
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发表时间：
1988
影响因子：
5.4
通讯作者：
Deepak Bastia
中科院分区：
文献类型：
--
作者：
Christopher A. Moskaluk;Deepak Bastia
牛乳头瘤病毒1型E2基因对位于病毒基因组长控制区的转录增强子具有正、负调控作用。E2基因产物的DNA结合域被怀疑与DNA序列基序ACCN 6 GGT相互作用。我们已经证明，E2蛋白的羧基末端126个氨基酸构成DNA结合结构域。本文报道了E2羧基端在大肠杆菌中的表达及其纯化。我们提供了明确的证据表明，该蛋白质识别的ACCN 6 GGT基序的病毒增强子。我们通过甲基化保护、甲基化干扰和乙基化干扰表明，E2蛋白在两条DNA链上的共有序列的GG残基处与DNA接触。凝胶阻滞-DNA酶I足迹测定显示，E2 DNA结合结构域对不同的ACCN 6 GGT基序表现出不同的亲和力，表明保守的ACC和GGT序列以外的核苷酸可能调节DNA序列对E2蛋白的亲和力。
The E2 gene of bovine papillomavirus type 1 positively and negatively regulates the transcriptional enhancer located in the long control region of the viral genome. The DNA-binding domain of the E2 gene product was suspected to interact with the DNA sequence motif ACCN6GGT. We have shown that the carboxy-terminal 126 amino acids of the E2 protein constitute the DNA-binding domain. In this paper we described the expression of the E2 carboxy terminus in Escherichia coli and its subsequent purification. We provide definitive evidence that the protein recognizes the ACCN6GGT motifs in the viral enhancer. We show by methylation protection, methylation interference, and ethylation interference that the E2 protein contacts the DNA at the GG residues of the consensus sequence on both DNA strands. A gel retardation-DNase I footprint assay has revealed that the E2 DNA-binding domain exhibits different affinities for different ACCN6GGT motifs, indicating that nucleotides other than the conserved ACC and GGT sequences probably modulate the affinity of the DNA sequence for the E2 protein.