Ujvári A, Martin C T
Department of Chemistry, University of Massachusetts, Amherst 01003-4510, USA.
Biochemistry. 1996 Nov 19;35(46):14574-82. doi: 10.1021/bi961165g.
Previous steady state kinetic studies of the initiation of transcription by T7 RNA polymerase have shown that melting of the DNA helix near the transcription start site is not rate limiting [Maslak, M., & Martin, C. T. (1993) Biochemistry 32, 4281-4285]. In the current work, fluorescence changes in a nucleotide analog incorporated within the promoter are used to monitor changes in the DNA helix associated with polymerase binding. The fluorescence of 2-aminopurine has been previously shown to depend on the environment of the base, with fluorescence increasing in the transition from a double-stranded to a single-stranded environment [Xu, D., Evans, K.O., & Nordlund, T. M.(1994) Biochemistry 33, 9592-9599]. Fluorescence changes associated with polymerase binding to promoters incorporating 2-aminopurine at positions -4 through -1 support a model which includes melting, in the statically bound complex, of the region of the promoter near the start site. Equilibrium titrations at 25 degrees C with label at position -2 provide a thermodynamic measure of the dissociation constant (Kd = 4.8 nM) for promoter binding, while stopped-flow kinetic assays measure the apparent association (k1 = 5.6 x 10(7) M-1 s-1) and dissociation (k-1 = 0.20 s-1) rate constants for simple promoter binding (the ratio k-1/k1 = 3.6 nM, in good agreement with the thermodynamic measurement of Kd). These results suggest that binding is close to the diffusion-controlled limit and helix melting is extremely rapid. In studies of structurally altered promoters, a base functional group substitution at position -10 is shown to significantly decrease k1, with little effect on k-1. In contrast, removal of the nontemplate strand from position +1 downstream results in a large decrease in k-1, with no significant effect on k1.
此前对T7 RNA聚合酶起始转录的稳态动力学研究表明，转录起始位点附近DNA螺旋的解旋并非限速步骤[马斯拉克，M.，&马丁，C. T.（1993年）《生物化学》32卷，4281 - 4285页]。在当前工作中，掺入启动子内的核苷酸类似物的荧光变化被用于监测与聚合酶结合相关的DNA螺旋变化。先前已表明2 - 氨基嘌呤的荧光取决于碱基所处环境，从双链环境转变为单链环境时荧光增强[徐，D.，埃文斯，K. O.，&诺德伦德，T. M.（1994年）《生物化学》33卷，9592 - 9599页]。与聚合酶结合到在 - 4至 - 1位置掺入2 - 氨基嘌呤的启动子相关的荧光变化支持了一个模型，该模型包括在静态结合复合物中起始位点附近启动子区域的解旋。在25℃下用位于 - 2位置的标记物进行平衡滴定，可得到启动子结合解离常数（Kd = 4.8 nM）的热力学测量值，而停流动力学分析则测量简单启动子结合的表观缔合速率常数（k1 = 5.6×10⁷ M⁻¹ s⁻¹）和解离速率常数（k⁻¹ = 0.20 s⁻¹）（k⁻¹/k1的比值为3.6 nM，与Kd的热力学测量值吻合良好）。这些结果表明结合接近扩散控制极限，且螺旋解旋极其迅速。在对结构改变的启动子的研究中， - 10位置的碱基官能团取代显示会显著降低k1，对k⁻¹影响较小。相反，从 + 1下游位置去除非模板链会导致k⁻¹大幅降低，对k1无显著影响。
