Jia Y, Kumar A, Patel S S
Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
J Biol Chem. 1996 Nov 29;271(48):30451-8. doi: 10.1074/jbc.271.48.30451.
The mechanism of bacteriophage T7 RNA polymerase binding to its promoter DNA was investigated using stopped-flow and equilibrium methods. To measure the kinetics of protein-DNA interactions in real time, changes in tryptophan fluorescence in the polymerase and 2-aminopurine (2-AP) fluorescence in the promoter DNA upon binary complex formation were used as probes. The protein fluorescence changes measured conformational changes in the polymerase whereas the fluorescence changes of 2-AP base, substituted in place of dA in the initiation region (-4 to +4), measured structural changes in the promoter DNA, such as DNA melting. The kinetic studies, carried out in the absence of the initiating nucleotide, are consistent with a two-step DNA binding mechanism, [formula: see text] where the RNA polymerase forms an initial weak EDa complex rapidly with an equilibrium association constant K1. The EDa complex then undergoes a conformational change to EDb, wherein RNA polymerase is specifically and tightly bound to the promoter DNA. Both the polymerase and the promoter DNA may undergo structural changes during this isomerization step. The isomerization of EDa to EDb is a fast step relative to the rate of transcription initiation and its rate does not limit transcription initiation. To understand how T7 RNA polymerase modulates its transcriptional efficiency at various promoters at the level of DNA binding, comparative studies with two natural T7 promoters, Phi10 and Phi3.8, were conducted. The results indicate that kinetics, the bimolecular rate constant of DNA binding, kon (K1k2), and the dissociation rate constant, koff (k-2), and thermodynamics, the equilibrium constants of the two steps (K1 and k2/k-2) both play a role in modulating the transcriptional efficiency at the level of DNA binding. Thus, the 2-fold lower kon, the 4-fold higher koff, and the 2-5-fold weaker equilibrium interactions together make Phi3.8 a weaker promoter relative to Phi10.
利用停流法和平衡法研究了噬菌体T7 RNA聚合酶与启动子DNA的结合机制。为实时测量蛋白质 - DNA相互作用的动力学，将聚合酶中色氨酸荧光的变化以及二元复合物形成时启动子DNA中2 - 氨基嘌呤（2 - AP）荧光的变化用作探针。测量的蛋白质荧光变化反映了聚合酶的构象变化，而取代起始区域（-4至+4）中dA的2 - AP碱基的荧光变化则测量了启动子DNA的结构变化，如DNA解链。在没有起始核苷酸的情况下进行的动力学研究与两步DNA结合机制一致，[公式：见原文]，其中RNA聚合酶以平衡缔合常数K1迅速形成初始弱EDa复合物。然后EDa复合物发生构象变化形成EDb，其中RNA聚合酶特异性且紧密地结合到启动子DNA上。在这个异构化步骤中，聚合酶和启动子DNA都可能发生结构变化。相对于转录起始速率，EDa到EDb的异构化是一个快速步骤，其速率并不限制转录起始。为了解T7 RNA聚合酶如何在DNA结合水平上调节其在各种启动子处的转录效率，对两个天然T7启动子Phi10和Phi3.8进行了比较研究。结果表明，动力学（DNA结合的双分子速率常数kon（K1k2）和解离速率常数koff（k - 2））以及热力学（两步的平衡常数（K1和k2 / k - 2））都在DNA结合水平上调节转录效率中发挥作用。因此，Phi3.8的kon低2倍、koff高4倍以及平衡相互作用弱2 - 5倍，使其相对于Phi10成为较弱的启动子。
