Bandwar R P, Patel S S
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
J Biol Chem. 2001 Apr 27;276(17):14075-82. doi: 10.1074/jbc.M011289200. Epub 2001 Jan 25.
The kinetics of promoter binding and open complex formation in bacteriophage T7 RNA polymerase was investigated using 2-aminopurine (2-AP) modified promoters. 2-AP serves as an ideal probe to measure the kinetics of open complex formation because its fluorescence is sensitive to both base-unpairing and base-unstacking and to the nature of the neighboring bases. All four 2-AP bases in the TATA box showed an increase in fluorescence with similar kinetics upon binding to the T7 RNA polymerase, indicating that the TATA sequence becomes unpaired in a concerted manner. The 2-AP at -4 showed a peculiarly large increase in fluorescence upon binding to the T7 RNA polymerase. Based on the recent crystal structure of the T7 RNA polymerase-DNA complex, we propose that the large fluorescence increase is due to unstacking of the 2-AP base at -4 from the guanine at -5, during open complex formation. The unstacking may be a critical event in directing and placing the template strand correctly in the T7 RNA polymerase active site upon promoter melting for template directed RNA synthesis. Based on equilibrium fluorescence and stopped-flow kinetic studies, we propose that a fast form of T7 RNA polymerase binds promoter double-stranded DNA by a three-step mechanism. The initial collision complex or a closed complex, ED(c) is formed with a K(d) of 1.8 microm. This complex isomerizes to an open complex, ED(o1), in an energetically unfavorable reaction with an equilibrium constant of 0.12. The ED(o1) further isomerizes to a more stable open complex, ED(o2), with a rate constant around 300 s(-1). Thus, in the absence of the initiating nucleotide, GTP, the overall equilibrium constant for closed to open complex conversion is 0.5 and the net rate of open complex formation is nearly 150 s(-1).
利用2-氨基嘌呤（2-AP）修饰的启动子研究了噬菌体T7 RNA聚合酶中启动子结合和开放复合物形成的动力学。2-AP是测量开放复合物形成动力学的理想探针，因为其荧光对碱基解配对、碱基解堆积以及相邻碱基的性质都很敏感。TATA框中的所有四个2-AP碱基在与T7 RNA聚合酶结合时，荧光都以相似的动力学增加，这表明TATA序列以协同方式解配对。位于-4位的2-AP在与T7 RNA聚合酶结合时荧光有特别大的增加。基于T7 RNA聚合酶-DNA复合物的最新晶体结构，我们提出这种大的荧光增加是由于在开放复合物形成过程中，-4位的2-AP碱基与-5位的鸟嘌呤解堆积所致。这种解堆积可能是在启动子解链以进行模板导向的RNA合成时，将模板链正确引导并放置在T7 RNA聚合酶活性位点的关键事件。基于平衡荧光和停流动力学研究，我们提出T7 RNA聚合酶的一种快速形式通过三步机制结合启动子双链DNA。最初形成碰撞复合物或封闭复合物ED(c)，其解离常数K(d)为1.8微摩尔。该复合物以能量不利的反应异构化为开放复合物ED(o1)，平衡常数为0.12。ED(o1)进一步异构化为更稳定的开放复合物ED(o2)，速率常数约为300 s⁻¹。因此，在没有起始核苷酸GTP的情况下，从封闭复合物到开放复合物转化的总体平衡常数为0.5，开放复合物形成的净速率接近150 s⁻¹。
