Greene R S, Munson B R
J Bacteriol. 1985 Jan;161(1):183-8. doi: 10.1128/jb.161.1.183-188.1985.
The interaction of RNA polymerase with a restriction fragment containing the origin of Escherichia coli replication (oriC) was examined by methods used to investigate transcription promoter activities. Interactions of RNA polymerase with oriC were determined and characterized by agarose gel exclusion under conditions of polymerase binding and RNA synthesis initiation. These interactions were further demonstrated and defined by nitrocellulose retention experiments under various reaction conditions. The binding of RNA polymerase to the oriC fragment was compared to binding to the tetracycline promoter (tet), a known strong promoter of transcription. Specific localization of the RNA polymerase-oriC interaction was determined by restriction protection experiments. The binding of RNA polymerase was determined to be located near the HindIII site of oriC. These methods allowed the observation and characterization of a specific association of RNA polymerase with the origin of E. coli DNA replication.
运用研究转录启动子活性的方法，对RNA聚合酶与含有大肠杆菌复制起点（oriC）的限制性片段之间的相互作用进行了研究。在聚合酶结合和RNA合成起始的条件下，通过琼脂糖凝胶排阻法测定并表征了RNA聚合酶与oriC的相互作用。在各种反应条件下，通过硝酸纤维素保留实验进一步证实并明确了这些相互作用。将RNA聚合酶与oriC片段的结合与已知的强转录启动子——四环素启动子（tet）的结合进行了比较。通过限制性保护实验确定了RNA聚合酶与oriC相互作用的特异性定位。确定RNA聚合酶的结合位于oriC的HindIII位点附近。这些方法使得对RNA聚合酶与大肠杆菌DNA复制起点之间特异性结合的观察和表征成为可能。
