Transient state kinetics of transcription elongation by T7 RNA polymerase
Transient state kinetics of transcription elongation by T7 RNA polymerase
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DOI：
10.1074/jbc.m608180200
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发表时间：
2006-11-24
影响因子：
4.8
通讯作者：
Patel, Smita S.
中科院分区：
文献类型：
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作者：
Anand, Vasanti Subramanian;Patel, Smita S.
来自噬菌体T7的单亚基dna依赖RNA聚合酶（RNAP）催化启动子依赖的转录起始和启动子独立的延伸。利用无启动子的底物，我们剖析了延伸过程中单核苷酸结合的动力学途径。我们发现T7 RNAP经历了一个缓慢的构象变化（0.01-0.03 s(-1)），与无启动子的底物（解离常数（K-d）为96 nM）形成了一个具有伸长能力的配合物。该复合物与正确的NTP （Kd为80 μ M）结合，并非常有效地将核苷单磷酸（NMP）结合到RNA引物中（在25℃下220 s(-1)）。根据测定的平衡常数，计算了单次NMP掺入的总自由能变化（-5.5 kcal/mol）和内部自由能变化（-3.7 kcal/mol）。在无机焦磷酸（PPi）存在下，延伸配合物催化反焦磷酸反应的最大速率为0.8 s(-1)， PPi K-d为1.2 mM。设计了几个实验来研究单核苷酸加成途径中的限速步骤。酸猝灭和脉冲追逐动力学表明，化学反应前的异构化步骤是限速的。两个核苷酸顺序结合的速率常数非常相似，表明化学反应后的步骤很快。基于现有的数据，我们提出在T7 RNAP的晶体学研究中观察到的NTP的插入前到插入异构化可能是限速步骤的候选。本研究为研究RNA合成的结构功能和保真度提供了一个动力学框架，并进一步探讨RNA合成过程中构象变化在核苷酸选择中的作用。
The single subunit DNA-dependent RNA polymerase ( RNAP) from bacteriophage T7 catalyzes both promoter-dependent transcription initiation and promoter-independent elongation. Using a promoter-free substrate, we have dissected the kinetic pathway of single nucleotide incorporation during elongation. We show that T7 RNAP undergoes a slow conformational change (0.01-0.03 s(-1)) to form an elongation competent complex with the promoter-free substrate ( dissociation constant (K-d) of 96 nM). The complex binds to a correct NTP ( Kd of 80 mu M) and incorporates the nucleoside monophosphate (NMP) into RNA primer very efficiently ( 220 s(-1) at 25 degrees C). An overall free energy change (-5.5 kcal/mol) and internal free energy change (-3.7 kcal/mol) of single NMP incorporation was calculated from the measured equilibrium constants. In the presence of inorganic pyrophosphate (PPi), the elongation complex catalyzes the reverse pyrophosphorolysis reaction at a maximum rate of 0.8 s(-1) with PPi K-d of 1.2 mM. Several experiments were designed to investigate the rate-limiting step in the pathway of single nucleotide addition. Acid-quench and pulse-chase kinetics indicated that an isomerization step before chemistry is rate-limiting. The very similar rate constants of sequential incorporation of two nucleotides indicated that the steps after chemistry are fast. Based on available data, we propose that the preinsertion to insertion isomerization of NTP observed in the crystallographic studies of T7 RNAP is a likely candidate for the rate-limiting step. The studies here provide a kinetic framework to investigate structure-function and fidelity of RNA synthesis and to further explore the role of the conformational change in nucleotide selection during RNA synthesis.