KINETICS OF OPEN COMPLEX-FORMATION BETWEEN ESCHERICHIA-COLI RNA-POLYMERASE AND THE LAC UV5 PROMOTER - EVIDENCE FOR A SEQUENTIAL MECHANISM INVOLVING 3 STEPS
KINETICS OF OPEN COMPLEX-FORMATION BETWEEN ESCHERICHIA-COLI RNA-POLYMERASE AND THE LAC UV5 PROMOTER - EVIDENCE FOR A SEQUENTIAL MECHANISM INVOLVING 3 STEPS
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DOI：
10.1021/bi00332a018
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发表时间：
1985-01-01
期刊：
影响因子：
2.9
通讯作者：
MCCLURE, WR
中科院分区：
文献类型：
--
作者：
BUC, H;MCCLURE, WR
研究了E.在15 - 42 ℃的温度范围内研究了大肠杆菌RNA聚合酶[RP]和lac UV 5启动子。C.标准的2步模型，涉及形成一个封闭的中间体，RPc，随后通过异构化，导致活性复合物RPo，不能占目前的数据。启动子-酶寿命测量显示出相反的温度依赖性（表观活化能，-35kcal/mol）。第三步，其与温度非常相关，并且在37 ℃下非常迅速。C，被假定为涉及DNA碱基对的解堆积，紧接着开放复合物的形成。将一个新的二元复合物，RPi，在该途径中的证据提供了由实验，区分稳定结合的物种和活性启动子后的温度跳跃扰动。这些实验允许测量稳定结合的物种之间的再平衡速率，并测定相应的平衡常数。第三步成为低于20 ℃的速率限制。C;这一预测是由正向动力学分析检查。在这3步模型中所涉及的参数的定量评价。在负超螺旋模板上进行了类似的实验：在这种情况下，即使在低温下，第三平衡也被驱动朝向开放复合物的形成，并且相应的步骤不是速率限制。
The forward and reverse kinetics of open complex formation between E. coli RNA polymerase [RP] and the lac UV5 promoter were studied in the temperature range of 15-42.degree. C. The standard 2-step model, involving the formation of a closed intermediate, RPc, followed by an isomerization that leads to the active complex RPo, could not account for the present data. The promoter-enzyme lifetime measurements showed an inverse temperature dependence (apparent activation energy, -35 kcal/mol). A 3rd step, which is very temperature dependent and which is very rapid at 37.degree. C, was postulated to involve the unstacking of DNA base pairs that immediately precedes open complex formation. Evidence for incorporating a new binary complex, RPi, in the pathway was provided by experiments that distinguished between stably bound species and active promoter after temperature-jump perturbations. These experiments allowed measurement of the rate of reequilibration between the stably bound species and determination of the corresponding equilibrium constant. The 3rd step became rate limiting below 20.degree. C; this prediction was checked by an analysis of the forward kinetics. A quantitative evaluation of the parameters involved in this 3-step model is provided. Similar experiments were performed on a negatively supercoiled template: in this case the 3rd equilibrium was driven toward formation of the open complex even at low temperature, and the corresponding step was not rate limiting.