KORB PROTEIN OF PROMISCUOUS PLASMID RP4 RECOGNIZES INVERTED SEQUENCE REPETITIONS IN REGIONS ESSENTIAL FOR CONJUGATIVE PLASMID TRANSFER
KORB PROTEIN OF PROMISCUOUS PLASMID RP4 RECOGNIZES INVERTED SEQUENCE REPETITIONS IN REGIONS ESSENTIAL FOR CONJUGATIVE PLASMID TRANSFER
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DOI：
10.1093/nar/20.8.1851
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发表时间：
1992-04-25
影响因子：
14.9
通讯作者：
LANKA, E
中科院分区：
文献类型：
--
作者：
BALZER, D;ZIEGELIN, G;LANKA, E
我们已经构建了RP 4科尔布高产菌株，并将蛋白纯化至接近同质。科尔布是一种DNA结合蛋白，识别确定的回文13-bp序列（TTTAGCSGCTAAA）。指定为O（B）的这种类型的反向序列重复出现在RP 4上12次。O（B）-序列位于复制和维持区以及接合转移所必需的Tral和Tra 2区。通过计算机搜索在Tra区域中发现的所有位点作为科尔布蛋白特异性结合的靶标。聚丙烯酰胺凝胶电泳DNA片段阻滞法检测KorB-DNA复合物。共有O（B）-序列的13-bp对称排列构成了结合科尔布蛋白的核心，因为该序列的任何截短都阻止复合物组装或导致KorB-DNA复合物的相当大的不稳定。羟基自由基足迹分析表明，复合物形成的科尔布与O（B）-序列直接，并建议一个不寻常的DNA结构内的核蛋白复合物的存在。
We have constructed a RP4 KorB overproducing strain and purified the protein to near homogeneity. KorB is a DNA binding protein recognizing defined palindromic 13-bp sequences (TTTAGCSGCTAAA). Inverted sequence repetitions of this type, designated O(B), are present on RP4 12 times. O(B)-sequences are localized in replication and maintenance regions as well as in the regions Tral and Tra2 essential for conjugative transfer. All sites found in Tra regions by computer search act as targets for specific binding of KorB protein. KorB-DNA complexes were detected by DNA fragment retardation assay using polyacrylamide gels. The 13-bp symmetric arrangement of the consensus O(B)-sequence constitutes the core for binding KorB protein since any truncation of this sequence prevents complex assembly or leads to a considerable destabilization of the KorB-DNA complexes. A hydroxyl radical footprint analysis demonstrated complex formation of KorB with the O(B)-sequence directly and suggests the presence of an unusual DNA structure within the nucleoprotein complex.