Kinetic mechanism of transcription initiation by bacteriophage T7 RNA polymerase
Kinetic mechanism of transcription initiation by bacteriophage T7 RNA polymerase
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DOI：
10.1021/bi9630467
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发表时间：
1997-04-08
期刊：
影响因子：
2.9
通讯作者：
Patel, SS
中科院分区：
文献类型：
--
作者：
Jia, YP;Patel, SS
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聚合酶的转录发生在三个阶段，包括起始、启动子清除和延伸。在起始阶段合成了6-8-mer的败育产物，从起始到延伸的过渡发生在6-8-mer和11-12-mer的合成之间，进行性延伸阶段在12-mer RNA合成之后开始。我们的研究结果表明，从Phi 10启动子的延伸产物的合成是有限的启动效率和频率的聚合酶逃逸的启动子。肝素陷阱的研究表明，聚合酶保持与启动子区域的流产RNA合成的多次周转期间的接触，因此，聚合酶不完全从启动子后，流产RNA合成的每个事件。RNA合成的前稳态动力学表明，起始发生的速率常数（3.5 s（-1））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主要受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的合成是不受限制的步骤与GTP结合，DNA结合，或启动子DNA的熔化。相反，动力学结果表明，在Phi 10启动子的起始受到第一个磷酸二酯键形成步骤的限制，或者更可能受到pppGpG形成之前的构象变化的限制。这样的构象变化可以发挥作用，在适当的启动和延长的NTP有效的磷酸二酯键的形成，并在保持RNA合成的保真度对齐。
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 Phi 10 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.