Purification and properties of the mini-F plasmid-encoded E protein needed for autonomous replication control of the plasmid.
Purification and properties of the mini-F plasmid-encoded E protein needed for autonomous replication control of the plasmid.
复制标题
质粒自主复制控制所需的 mini-F 质粒编码的 E 蛋白的纯化和特性。
DOI：
10.1073/pnas.83.12.4109
复制
发表时间：
1986
影响因子：
11.1
通讯作者：
K. Matsubara
中科院分区：
文献类型：
--
作者：
T. Tokino;T. Murotsu;K. Matsubara
Mini-F 质粒编码一种蛋白质，即 E 蛋白，这是其自主复制所必需的。我们构建了一种过量产生 E 蛋白的质粒，并将该蛋白纯化至明显的同质性。使用硝酸纤维素滤膜结合和核酸酶消化测定，我们证明E蛋白与mini-F DNA序列的三个独特区域结合：复制起点(ori2)和不相容性位点(incB)、另一个不相容性位点(incC)以及E基因的启动子。这些结合位点具有共同的 8 碱基对序列。这些发现表明 E 蛋白在 mini-F 复制起始和拷贝数控制中发挥直接作用。它们也与体内证据一致，即由 incB 和 incC DNA 引起的不相容表型是由于复制不可缺少的因子的滴定造成的，并且 mini-F 质粒的 E 起始蛋白的产生处于自动调节控制之下。
Mini-F plasmid encodes a protein, E protein, that is indispensable for its autonomous replication. We have constructed a plasmid that overproduces the E protein and have purified the protein to apparent homogeneity. Using nitrocellulose filter binding and nuclease digestion assays, we demonstrated that the E protein binds to three unique regions of the mini-F DNA sequence: the replication origin (ori2) and an incompatibility locus (incB), another incompatibility locus (incC), and the promoter for the E gene. These binding sites have a common 8-base-pair sequence. These findings suggest the direct role of the E protein in initiation of mini-F replication and copy number control. They are also in line with the in vivo evidence that the incompatibility phenotype caused by incB and incC DNA is due to titration of a factor(s) indispensable for replication and that the production of the E initiator protein of the mini-F plasmid is under autoregulatory control.