Craig M L, Tsodikov O V, McQuade K L, Schlax P E, Capp M W, Saecker R M, Record M T
Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
J Mol Biol. 1998 Nov 6;283(4):741-56. doi: 10.1006/jmbi.1998.2129.
Kinetic studies of formation and dissociation of open-promoter complexes (RPo) involving Esigma70 RNA polymerase (R) and the lambdaPR promoter (P) demonstrate the existence of two kinetically significant intermediates, designated I1 and I2, and facilitate the choice of conditions under which each accumulates. For such conditions, we report the results of equilibrium and transient DNase I and KMnO4 footprinting studies which characterize I1 and I2. At 0 degreesC, where extrapolation of equilibrium data indicates I1 is the dominant complex, DNA bases in the vicinity of the transcription start site (+1) do not react with KMnO4, indicating that this region is closed in I1. However, the DNA backbone in I1 is extensively protected from DNase I cleavage; the DNase I footprint extends approximately 30 bases downstream and at least approximately 40 bases upstream from the start site. I1 has a short lifetime (</=15 seconds), based on its sensitivity to competition with heparin. Shortly after a temperature downshift from 37 degreesC to 0 degreesC, in the time-range where we conclude that the dominant, transiently accumulated complex is I2, DNase I and KMnO4 footprinting reveal a complex with a closed-start site and an extended DNase I footprint like that of I1. However, unlike I1, I2 is insensitive to heparin competition and has a much longer dissociation lifetime at 0 degreesC. Based on footprinting, kinetic and thermodynamic studies, we conclude that in the short-lived intermediate I1 the promoter start site and downstream region are bound in a cleft defined by the open clamp-like jaws of Esigma70. We propose that binding of the start site and downstream DNA in this cleft triggers massive, relatively slow conformational changes which likely include RNA polymerase jaw closing with coupled folding. These proposed conformational changes occur prior to opening of the promoter start site region, and are responsible for the much longer lifetime of I2. Closing of the jaws of polymerase around the downstream region of promoter DNA appears to trigger opening of the start site region. From a quantitative analysis of the biphasic decay of KMnO4 reactivity of RPo at 0 degreesC, we obtain the equilibrium constant K3 for the conversion of I2 to RPo and the rate constant k-2 for the conversion of I2 to I1 (i.e. jaw opening). These quantitative results were previously unavailable at any temperature, and are necessary for the dissection of dissociation kinetic data at higher temperatures.
对涉及大肠杆菌σ⁷⁰ RNA聚合酶（R）和λPR启动子（P）的开放启动子复合物（RPo）形成和解离的动力学研究表明，存在两种具有动力学意义的中间体，分别命名为I1和I2，并有助于选择每种中间体积累的条件。针对这些条件，我们报告了平衡以及瞬时DNase I和高锰酸钾足迹分析研究的结果，这些研究对I1和I2进行了表征。在0℃时，平衡数据的外推表明I1是主要复合物，转录起始位点（+1）附近的DNA碱基不与高锰酸钾反应，这表明该区域在I1中是封闭的。然而，I1中的DNA主链受到广泛保护，免受DNase I切割；DNase I足迹从起始位点向下游延伸约30个碱基，向上游至少延伸约40个碱基。基于其对肝素竞争的敏感性，I1的寿命较短（≤15秒）。在温度从37℃迅速下降到0℃后不久，在我们得出主要的瞬时积累复合物是I2的时间范围内，DNase I和高锰酸钾足迹分析揭示了一种具有封闭起始位点和与I1类似的扩展DNase I足迹的复合物。然而，与I1不同，I2对肝素竞争不敏感，并且在0℃时具有长得多的解离寿命。基于足迹分析、动力学和热力学研究，我们得出结论，在寿命短暂的中间体I1中，启动子起始位点和下游区域结合在由大肠杆菌σ⁷⁰的开放钳状结构域定义的裂隙中。我们提出，起始位点和下游DNA在该裂隙中的结合触发了大量相对缓慢的构象变化，这可能包括RNA聚合酶结构域闭合并伴随折叠。这些提出的构象变化发生在启动子起始位点区域开放之前，并导致I2的寿命长得多。聚合酶围绕启动子DNA下游区域的结构域闭合似乎触发了起始位点区域的开放。通过对0℃下RPo的高锰酸钾反应性双相衰减的定量分析，我们获得了I2转化为RPo的平衡常数K3以及I2转化为I1（即结构域开放）的速率常数k-2。这些定量结果在任何温度下以前都无法获得，并且对于剖析更高温度下的解离动力学数据是必要的。
