McClure W R
Proc Natl Acad Sci U S A. 1980 Oct;77(10):5634-8. doi: 10.1073/pnas.77.10.5634.
Promoter-specific lags in the approach to the steady-state rate of abortive initiation were observed when Escherichia coli RNA polymerase was added to initiate the reaction. The lag times were related to the time required for free enzyme and free promoter to combine and isomerize into a functionally active complex. The lag times measured for several bacteriophage and bacterial promoters differed widely (10 sec to several minutes) and in most cases corresponded to the rate-limiting step in the initiation process. The unique advantage in using the abortive initiation reaction to measure the lags was that the binding and isomerization steps in a simple two-state model could be quantitated separately. The separation of the contributions of both steps was effected by deriving an equation to describe the rate of formation of the active binary complex. Results from experiments based on the theory showed a linear relationship between the observed lag times and the reciprocal enzyme concentration. The slope and intercept of the equation yielded quantitative estimates of the binding and isomerization steps in initiation. The analysis was applied to the bacteriophage T7 A2 and D promoters to show the bases for the differences in in vitro initiation frequency that have been observed for these promoters.
当加入大肠杆菌RNA聚合酶来启动反应时，观察到在趋向流产起始稳态速率的过程中存在启动子特异性延迟。延迟时间与游离酶和游离启动子结合并异构化为功能活性复合物所需的时间有关。对几种噬菌体和细菌启动子测量的延迟时间差异很大（10秒到几分钟），并且在大多数情况下对应于起始过程中的限速步骤。使用流产起始反应来测量延迟的独特优势在于，简单双态模型中的结合和异构化步骤可以分别进行定量。通过推导一个描述活性二元复合物形成速率的方程，实现了这两个步骤贡献的分离。基于该理论的实验结果表明，观察到的延迟时间与酶浓度的倒数之间存在线性关系。该方程的斜率和截距给出了起始中结合和异构化步骤的定量估计。该分析应用于噬菌体T7 A2和D启动子，以揭示这些启动子在体外起始频率上差异的基础。
