Jia Y, Patel S S
Department of Biochemistry, The Ohio State University, Columbus 43210, USA.
Biochemistry. 1997 Apr 8;36(14):4223-32. doi: 10.1021/bi9630467.
The kinetic mechanism of transcription initiation by bacteriophage T7 RNA polymerase was investigated using transient state kinetic methods. Transcription by bacteriophage T7 RNA polymerase occurs in three stages consisting of initiation, promoter clearance, and elongation. Abortive products, up to 6-8-mer, were synthesized during the initiation phase; the transition from initiation to elongation occurred between the synthesis of 6-8-mer and 11-12-mer, and the processive elongation phase began after the synthesis of 12-mer RNA. Our results show that the synthesis of elongation product from the phi 10 promoter is limited both by the efficiency of initiation and by the frequency at which the polymerase escapes the promoter. Studies with heparin trap suggest that the polymerase maintains contact with the promoter region during multiple turnovers of abortive RNA synthesis; thus, the polymerase does not completely dissociate from the promoter after each event of abortive RNA synthesis. The pre-steady-state kinetics of RNA synthesis indicate that initiation occurs at a rate constant (3.5 s(-1)) that is about 30 times faster than the steady-state rate constant of RNA synthesis (0.1 s(-1)). The steady-state rate constant of RNA synthesis is limited largely by the cycling of the RNA polymerase, whereas initiation is limited by the formation of pppGpG, the first RNA product. We show that the synthesis of pppGpG is not limited by steps associated with GTP binding, DNA binding, or the melting of the promoter DNA. Instead, the kinetic results indicate that initiation at the phi10 promoter is limited either by the first phosphodiester bond formation step or more likely by a conformational change prior to pppGpG formation. Such a conformational change could play a role in proper alignment of the initiating and elongating NTPs for efficient phosphodiester bond formation and in maintaining the fidelity of RNA synthesis.
利用瞬态动力学方法研究了噬菌体T7 RNA聚合酶转录起始的动力学机制。噬菌体T7 RNA聚合酶的转录过程包括起始、启动子清除和延伸三个阶段。在起始阶段合成了长度达6 - 8聚体的流产产物；从起始到延伸的转变发生在6 - 8聚体和11 - 12聚体合成之间，而连续延伸阶段在12聚体RNA合成之后开始。我们的结果表明，从phi 10启动子合成延伸产物既受起始效率的限制，也受聚合酶逃离启动子频率的限制。肝素捕获实验表明，在流产RNA合成的多次周转过程中，聚合酶与启动子区域保持接触；因此，在每次流产RNA合成事件后，聚合酶不会完全从启动子上解离。RNA合成的预稳态动力学表明，起始以速率常数（3.5 s(-1)）发生，该速率常数比RNA合成的稳态速率常数（0.1 s(-1)）快约30倍。RNA合成的稳态速率常数在很大程度上受RNA聚合酶循环的限制，而起始则受第一个RNA产物pppGpG形成的限制。我们表明，pppGpG的合成不受与GTP结合、DNA结合或启动子DNA解链相关步骤的限制。相反，动力学结果表明，在phi10启动子处的起始要么受第一个磷酸二酯键形成步骤的限制，更有可能受pppGpG形成之前的构象变化的限制。这种构象变化可能在起始和延伸的NTP正确排列以实现高效磷酸二酯键形成以及维持RNA合成保真度方面发挥作用。
