Cook Victoria M, Dehaseth Pieter L
Center for RNA Molecular Biology, Case Western Reserve University, Clevland, Ohio 44106, USA.
J Biol Chem. 2007 Jul 20;282(29):21319-26. doi: 10.1074/jbc.M702232200. Epub 2007 May 16.
Formation of the strand-separated, open complex between RNA polymerase and a promoter involves several intermediates, the first being the closed complex in which the DNA is fully base-paired. This normally short lived complex has been difficult to study. We have used a mutant Escherichia coli RNA polymerase, deficient in promoter DNA melting, and variants of the P(R) promoter of bacteriophage lambda to model the closed complex intermediate at physiologically relevant temperatures. Our results indicate that in the closed complex, RNA polymerase recognizes base pairs as double-stranded DNA even in the region that becomes single-stranded in the open complex. Additionally, a particular base pair in the -35 region engages in an important interaction with the RNA polymerase, and a DNase I-hypersensitive site, pronounced in the promoter DNA of the open complex, was not present. The effect of temperature on closed complex formation was found to be small over the temperature range from 15 to 37 degrees C. This suggests that low temperature complexes of wild type RNA polymerase and promoter DNA may adequately model the closed complex.
RNA聚合酶与启动子之间形成链分离的开放复合物涉及多个中间体，第一个是封闭复合物，其中DNA完全碱基配对。这个通常寿命短暂的复合物一直难以研究。我们使用了一种在启动子DNA解链方面存在缺陷的突变型大肠杆菌RNA聚合酶，以及噬菌体λ的P(R)启动子变体，在生理相关温度下模拟封闭复合物中间体。我们的结果表明，在封闭复合物中，RNA聚合酶即使在开放复合物中变为单链的区域也将碱基对识别为双链DNA。此外，-35区域的一个特定碱基对与RNA聚合酶发生重要相互作用，并且在开放复合物的启动子DNA中明显的DNase I超敏感位点并不存在。发现在15至37摄氏度的温度范围内，温度对封闭复合物形成的影响很小。这表明野生型RNA聚合酶与启动子DNA的低温复合物可能足以模拟封闭复合物。
