DNA footprints of the two kinetically significant intermediates in formation of an RNA polymerase-promoter open complex: Evidence that interactions with start site and downstream DNA induce sequential conformational changes in polymerase and DNA
DNA footprints of the two kinetically significant intermediates in formation of an RNA polymerase-promoter open complex: Evidence that interactions with start site and downstream DNA induce sequential conformational changes in polymerase and DNA
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DOI：
10.1006/jmbi.1998.2129
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发表时间：
1998-11-06
影响因子：
5.6
通讯作者：
Record, MT
中科院分区：
文献类型：
--
作者：
Craig, ML;Tsodikov, OV;Record, MT
对涉及E sigma（70）RNA聚合酶（R）和λ P-R启动子（P）的开放启动子复合物（RP）的形成和解离的动力学研究表明，存在两种动力学上重要的中间体，命名为I-1和I-2，并有助于选择每种累积的条件。对于这样的条件下，我们报告的结果，平衡和瞬态DNase I和KMnO 4足迹的研究，其特征I-1和I-2。在0 ℃时，平衡数据的外推表明I-1是主要复合物，转录起始位点（+1）附近的DNA碱基不与KMnO 4反应，表明该区域在I-1中是封闭的。然而，I中的DNA骨架受到广泛保护，免受DNase I切割; DNase I足迹延伸至起始位点下游30个碱基，至少延伸至起始位点上游40个碱基。基于其对肝素竞争的敏感性，I具有短的寿命（小于或等于15秒）。在温度从37摄氏度降到0摄氏度后不久，在我们得出结论的时间范围内，占主导地位的，短暂积累的复合物：是I-2，DNA酶I和KMnO 4足迹揭示了一个复杂的封闭的起始位点和扩展的DNA酶I足迹一样的I-1。然而，与I-1不同，I-2对肝素竞争不敏感，并且在0摄氏度下具有长得多的解离寿命。基于足迹法、动力学和热力学研究，我们得出结论，在短寿命的中间体I-1中，启动子起始位点和下游区域结合在由E sigma的开放钳状钳口限定的裂缝中（70）。我们建议，在这个clef的起始位点和下游DNA的结合触发大规模的，相对缓慢的构象变化，其中可能包括RNA聚合酶颚关闭与耦合折叠。这些建议的构象变化发生在启动子起始位点区域的开放之前，并且负责I-2的更长的寿命。启动子DNA下游区域周围聚合酶钳口的闭合似乎触发起始位点区域的打开。从定量分析的双相衰减的KMnO_4反应性的RPo，在0 ℃下，我们得到的平衡常数K-3的转换的I-2到RPo和转换的I-2到I-1（即颚打开）的速率常数k（-2）。这些定量结果以前在任何温度下都是不可用的，并且对于在更高温度下分解动力学数据是必要的。(C)北京：科学出版社.
Kinetic studies of formation and dissociation of open-promoter complexes (RP,) involving E sigma(70) RNA polymerase (R) and the lambda P-R promoter (P) demonstrate the existence of two kinetically significant intermediates, designated I-1 and I-2, 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 I-1 and I-2. At 0 degrees C, where extrapolation of equilibrium data indicates I-1 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 I-1. However, the DNA backbone in I, is extensively protected from DNase I cleavage; the DNase I footprint extends similar to 30 bases downstream and at least similar to 40 bases upstream from the start site. I, has a short lifetime (less than or equal to 15 seconds), based on its sensitivity to competition with heparin. Shortly after a temperature downshift from 37 degrees C to 0 degrees C, in the time-range where we conclude that the dominant, transiently accumulated complex:is I-2,DNase I and KMnO4 footprinting reveal a complex with a closed-start site and an extended DNase I footprint like that of I-1. However, unlike I-1, I-2 is insensitive to heparin competition and has a much longer dissociation lifetime at 0 degrees C.Based on footprinting, kinetic and thermodynamic studies, we conclude that in the short-lived intermediate I-1 the promoter start site and downstream region are bound in a cleft defined by the open clamp-like jaws of E sigma(70). We propose that binding of the start site and downstream DNA in this clef 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 I-2. 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 degrees C, we obtain the equilibrium constant K-3 for the conversion of I-2 to RPo and the rate constant k(-2) for the conversion of I-2 to I-1 (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. (C) 1998 Academic Press.