DNA-protein interaction at the replication origins of plasmid chromosomes.
DNA-protein interaction at the replication origins of plasmid chromosomes.
复制标题
质粒染色体复制起点处的 DNA-蛋白质相互作用。
DOI：
10.1007/978-1-4613-2447-8_29
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
Gray,J
中科院分区：
文献类型：
--
作者：
Bastia,D;Vocke,C;Germino,J;Gray,J
研究了纯化质粒R6K和pSC101复制启动蛋白的新技术。该技术包括在正确的翻译阶段用β-半乳糖苷酶编码大肠杆菌DNA标记c端启动子。然后通过吸附和从β-半乳糖苷酶特异性亲和柱洗脱快速纯化杂交蛋白。已经设计了两种程序来分离未融合的引发蛋白，使用融合蛋白作为手柄。第一种方法称为亚单位关联层析，利用β-半乳糖苷酶特异性亲和柱层析分离非标记蛋白单体与β-半乳糖苷酶标记蛋白单体的关联。第二步是通过特定的连接体DNA将引发蛋白与β-半乳糖苷酶融合。连接体DNA编码一种蛋白质，该蛋白质容易被序列特异性蛋白酶特异性水解，从而从β-半乳糖苷酶释放引发蛋白。使用纯化或部分纯化的启动蛋白，我们已经证明R6K编码的启动蛋白（Pi蛋白）与质粒染色体2个区域的22 bp序列一致结合。psc101编码的启动蛋白与质粒复制起点或附近的序列结合。在低浓度下，蛋白质与成核位点结合，当提高浓度时，蛋白质与成核序列部分同源的4个相邻序列的结合被促进。结合位点的删除会导致非功能性复制起点。
Novel techniques have been developed to purify replication initiator proteins of the plasmids R6K and pSC101. The techniques consist of tagging the initiator cistrons at the C-terminus with β-galactosidase-encoding DNA ofEscherichiacoliin the correct translational phase. The hybrid proteins are then rapidly purified by adsorption to and elution from a β-galactosidase-specific affinity column. Two procedures have been devised to isolate the nonfused initiator proteins using the fused protein as a handle. The first procedure, called subunit association chromatography, exploits the association of a monomer of nontagged protein with that of β-galactosidase-tagged protein in isolating both types of proteins by β-galactosidase specific affinity column chromatography. The second procedure involves the fusion of the initiator protein to β-galactosidase via a specific linker DNA. The linker DNA encodes a protein which is readily and specifically hydrolyzed by a sequence specific protease, thus releasing the initiator protein from β-galactosidase. Using purified or partially purified initiator protein, we have demonstrated that the R6K encoded initiator protein (Pi protein) binds to a consensus 22 bp sequence at 2 regions of the plasmid chromosome. The pSC101-encoded initiator protein binds to sequences at or near the plasmid replication origin. At low concentrations the protein binds to a nucleation site and upon raising the concentrations of the protein binding is promoted at 4 adjacent sequences that have partial homologies with the nucleation sequence. Deletion of the binding site leads to a nonfunctional replication origin.