Prosen D E, Cech C L
Biochemistry. 1986 Sep 23;25(19):5378-87. doi: 10.1021/bi00367a005.
A specific Escherichia coli RNA polymerase tight-binding (TB) site on bacteriophage T7 has been located at 32,988 base pairs from the left end of T7. This site is referred to as the T7 F promoter since it is fully active in vitro. Kinetics of association and dissociation have been measured by use of the abortive initiation assay and runoff transcription. The association constant, ka approximately 9 X 10(5) M-1 s-1, is of the same magnitude as ka for the T7 minor promoters. In competitive titration assays, the F promoter was found to be slightly weaker than the minor T7 E promoter at low RNA polymerase concentrations and, as expected, much weaker than the major T7 A3 promoter. An unusual RNA polymerase mediated inhibition of both the association rate and the transcriptional activity was observed at moderately high concentrations of polymerase. A mechanistic model analogous to enzyme substrate inhibition is presented.
噬菌体T7上一个特定的大肠杆菌RNA聚合酶紧密结合（TB）位点位于距T7左端32,988个碱基对处。该位点被称为T7 F启动子，因为它在体外具有完全活性。通过使用流产起始测定法和连续转录法测量了结合和解离动力学。结合常数ka约为9×10⁵ M⁻¹ s⁻¹，与T7次要启动子的ka大小相同。在竞争性滴定测定中，发现在低RNA聚合酶浓度下，F启动子比次要的T7 E启动子略弱，并且正如预期的那样，比主要的T7 A3启动子弱得多。在中等高浓度的聚合酶下，观察到RNA聚合酶对结合速率和转录活性均有异常抑制作用。提出了一种类似于酶底物抑制的机制模型。
