Kolasa I K, Loziński T, Wierzchowski K L
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
Acta Biochim Pol. 2001;48(4):985-94.
The kinetics and thermodynamics of the formation of the transcriptional open complex (RPo) by Escherichia coli RNA polymerase at the synthetic Pa promoter bearing consensus -10 and -35 recognition hexamers were studied in vitro. Previously, this promoter was used as a control one in studies on the effect of DNA bending by An x Tn sequences on transcription initiation and shown to be fully functional in E. coli (Loziński et al., 1991, Nucleic Acids Res. 19, 2947; Loziński & Wierzchowski, 1996, Acta Biochim. Polon. 43, 265). The data now obtained demonstrate that the mechanism of Pa-RPo formation and dissociation conforms to the three-step reaction model: bind-nucleate-melt, commonly accepted for natural promoters. Measurements of the dissociation rate constant of Pa-RPo as a function of MgCl2 concentration allowed us to determine the number of Mg2+ ions, nMg approximately/= 4, being bound to the RPo in the course of renaturation of the melted DNA region. This number was found constant in the temperature range of 25-37 degrees C, which indicates that under these conditions the complex remaines fully open. This observation, taken together with the recent evidence from KMnO4 footprinting studies that the length of the melted region in Pa-RPo at 37 degrees C is independent of the presence of Mg2+ ions (Lozinski & Wierzchowski, 2001, Acta Biochim. Polon. 48, 495), testifies that binding of Mg2+ to RPo does not induce its further isomerization, which has been postulated for the lambdaP(R)-RPo complex (Suh et al., 1992, Biochemistry 31, 7815; 1993, Science 259, 358).
在体外研究了大肠杆菌RNA聚合酶在带有共有-10和-35识别六聚体的合成Pa启动子处形成转录开放复合物（RPo）的动力学和热力学。此前，在研究An x Tn序列引起的DNA弯曲对转录起始的影响时，该启动子被用作对照启动子，并已证明其在大肠杆菌中具有完全功能（Loziński等人，1991年，《核酸研究》19卷，2947页；Loziński和Wierzchowski，1996年，《波兰生物化学学报》43卷，265页）。现在获得的数据表明，Pa-RPo形成和解离的机制符合三步反应模型：结合-成核-解链，这是天然启动子普遍接受的模型。测量Pa-RPo的解离速率常数随MgCl2浓度的变化，使我们能够确定在熔化的DNA区域复性过程中与RPo结合的Mg2+离子数量，nMg约为4。在25-37摄氏度的温度范围内，该数量保持恒定，这表明在这些条件下复合物保持完全开放。这一观察结果，结合最近来自高锰酸钾足迹研究的证据，即在37摄氏度时Pa-RPo中熔化区域的长度与Mg2+离子的存在无关（Lozinski和Wierzchowski，2001年，《波兰生物化学学报》48卷，495页），证明Mg2+与RPo的结合不会诱导其进一步异构化，而λP（R）-RPo复合物曾被假定存在这种异构化（Suh等人，1992年，《生物化学》31卷，7815页；1993年，《科学》259卷，358页）。
