Swack J A, Pal S K, Mason R J, Abeles A L, Chattoraj D K
J Bacteriol. 1987 Aug;169(8):3737-42. doi: 10.1128/jb.169.8.3737-3742.1987.
To study the functions of the mini-P1 replication initiation protein RepA quantitatively, we have developed a method to measure RepA concentration by using immunoblotting. In vivo, there are about 20 RepA dimers per unit-copy plasmid DNA. RepA was deduced to be a dimer from gel filtration of the purified protein. Since there are 14 binding sites of the protein per replicon, the physiological concentration of the protein appears to be sufficiently low to be a rate-limiting factor for replication. Autoregulation is apparently responsible for the low protein level; at the physiological concentration of the protein, the repA promoter retains only 0.1% of its full activity as determined by gene fusions to lacZ. When the concentration is further decreased by a factor of 3 or increased by a factor of 40, replication is no longer detectable.
为了定量研究微型P1复制起始蛋白RepA的功能，我们开发了一种利用免疫印迹法测量RepA浓度的方法。在体内，每单位拷贝的质粒DNA约有20个RepA二聚体。通过对纯化蛋白进行凝胶过滤，推断RepA为二聚体。由于每个复制子有该蛋白的14个结合位点，该蛋白的生理浓度似乎足够低，足以成为复制的限速因子。自我调节显然是导致蛋白水平较低的原因；在该蛋白的生理浓度下，通过与lacZ的基因融合测定，repA启动子仅保留其全部活性的0.1%。当浓度进一步降低3倍或增加40倍时，不再能检测到复制。
