Characterization of the DNA binding domain of bacteriophage lambda O protein.
Characterization of the DNA binding domain of bacteriophage lambda O protein.
复制标题
噬菌体 lambda O 蛋白 DNA 结合域的表征。
DOI：
10.1016/s0021-9258(17)38425-9
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
K. Zahn
中科院分区：
文献类型：
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作者：
S. Wickner;K. Zahn
O和P基因产物是启动DNA复制所必需的。为了研究这一过程的生物化学，我们构建了携带lambda O基因、P基因和编码O蛋白氨基末端一半的O基因的质粒。每一种蛋白都受可诱导的λ启动子PL控制。我们从携带质粒的诱导细胞中纯化了这三种蛋白。我们的研究结果表明，O蛋白的氨基末端部分以类似于完整的lambda O蛋白的方式结合到复制的lambda原点，这表明O蛋白的氨基末端部分包含DNA结合域。使用色谱程序，我们已经分离了一个复合物的λ O和P蛋白与λ dv DNA。在相同条件下，O蛋白的氨基末端部分不与P蛋白络合。这表明O蛋白对P蛋白的特异性存在于O蛋白的羧基末端。我们的研究结果还表明，虽然完整的O蛋白在lambda dv质粒DNA的体外复制中具有活性，但O蛋白的氨基末端部分是无活性的，并且在该反应中是lambda O蛋白的竞争性抑制剂。这些结果证实了先前的遗传观察结果，即lambda O蛋白具有双功能结构，其中氨基末端结构域识别lambda复制的起源，羧基末端结构域与lambda P蛋白相互作用。
The lambda O and P gene products are required for the initiation of lambda DNA replication. In order to study the biochemistry of this process, we have constructed plasmids that carry the lambda O gene, P gene, and half of the O gene coding for the amino-terminal half of the O protein. Each is under the control of the inducible lambda promoter, PL. We have purified these three proteins from induced cells carrying the plasmids. Our results show that the amino-terminal portion of the O protein binds to the lambda origin of replication in a manner similar to the intact lambda O protein, demonstrating that the amino-terminal portion of O protein contains the DNA binding domain. Using chromatographic procedures, we have isolated a complex of lambda O and P proteins with lambda dv DNA. The amino-terminal portion of the O protein does not complex with P protein under the same conditions. This suggests that the specificity of the lambda O protein for P protein resides in the carboxyl-terminal half of the lambda O protein. Our results also show that, while the intact O protein is active in in vitro replication of lambda dv plasmid DNA, the amino-terminal portion of the O protein is inactive and is a competitive inhibitor of the lambda O protein in this reaction. These results confirm previous genetic observations that were interpreted as indicating a bifunctional structure for the lambda O protein with the amino-terminal domain recognizing the lambda origin of replication and the carboxyl-terminal domain interacting with the lambda P protein.