Abstract:
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).
摘要:
体外研究了大肠杆菌RNA聚合酶在合成的Pa启动子上形成转录开放复合物(RPo)的动力学和热力学,该启动子具有共有-10和-35识别六聚体。以前,在研究AnxTn序列对DNA弯曲对转录起始的影响的研究中,该启动子被用作对照,并在大肠杆菌中显示出完全的功能(Loziñski等人。,1991年,核酸研究。19,2947;洛辛斯基和维尔茨乔夫斯基,1996年,ActaBiochim。波隆.43,265)。现在获得的数据表明,Pa-RPo形成和解离的机理符合三步反应模型:结合-成核-熔体,通常被接受为天然启动子。测量Pa-RPo的解离速率常数与MgCl2浓度的关系,使我们能够确定Mg2离子的数量,nMg大约=4,在解链DNA区域复性过程中与RPo结合。这个数字在25-37摄氏度的温度范围内是恒定的,这表明在这些条件下,建筑群仍然完全开放。这个观察,与KMnO4足迹研究的最新证据一起考虑,在37摄氏度下Pa-RPo中熔化区域的长度与Mg2离子的存在无关(Lozinski&Wierzchowski,2001年,ActaBiochim。波隆.48,495),证明Mg2+与RPo的结合不会诱导其进一步异构化,这已经被假定为lambdaP(R)-RPo复合物(Suh等人。,1992,生物化学31,7815;1993,科学259,358)。