von Gabain A, Bujard H
Proc Natl Acad Sci U S A. 1979 Jan;76(1):189-93. doi: 10.1073/pnas.76.1.189.
The interaction of Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) with restriction fragments obtained from various E. coli related DNAs was studied in vitro. The DNAs investigated included several coliphage genomes (T5, lambda, T7, fd) and plasmid DNAs (pML 21, pSC101). By using the nitrocellulose filter binding of the enzyme-DNA complexes, fragment-specific relative rates of complex formation as well as complex stabilities were determined. Promoter-specific relative rates of polymerase binding were derived from fragment-specific rates by taking into account the number of major binding sites for RNA polymerase within several DNAs. Estimates of the stability of complexes formed between some major binding sites and the enzyme were obtained by studying the rate of complex decay. Both characteristics--rate of complex formation and rate of decay--varied widely and independently of each other. The promoters reacting most efficiently with E. coli RNA polymerase were found in the early region of coliphage T5 whereas some promoters in pML 21, or for example, the lambda promoter PI, belong to signals binding the enzyme most slowly. Based on the second-order rate constant determined for the interaction of E. coli RNA polymerase with promoters of phage fd, the fastest promoters characterized so far reacted with rates in the order of 10(8) M-1s-1. The hierarchy of promoters established here is of interest from the viewpoint that promoter strength correlates with the rate of polymerase binding. Among the promoters studied here this rate spans a range of 2 orders of magnitude.
在体外研究了大肠杆菌RNA聚合酶（核苷三磷酸：RNA核苷酸转移酶，EC 2.7.7.6）与从各种大肠杆菌相关DNA获得的限制性片段的相互作用。所研究的DNA包括几种大肠杆菌噬菌体基因组（T5、λ、T7、fd）和质粒DNA（pML 21、pSC101）。通过使用酶-DNA复合物的硝酸纤维素滤膜结合法，测定了片段特异性的复合物形成相对速率以及复合物稳定性。通过考虑几种DNA中RNA聚合酶的主要结合位点数量，从片段特异性速率推导出启动子特异性的聚合酶结合相对速率。通过研究复合物衰变速率，获得了一些主要结合位点与酶形成的复合物稳定性的估计值。复合物形成速率和衰变速率这两个特征变化很大，且彼此独立。发现与大肠杆菌RNA聚合酶反应最有效的启动子位于噬菌体T5的早期区域，而pML 21中的一些启动子，例如λ启动子P1，属于与酶结合最慢的信号。根据为大肠杆菌RNA聚合酶与噬菌体fd启动子相互作用测定的二级速率常数，迄今为止表征的最快启动子的反应速率约为10(8) M-1s-1。从启动子强度与聚合酶结合速率相关的观点来看，这里建立的启动子层级很有趣。在这里研究的启动子中，这个速率跨度为2个数量级。
