Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University, Greenville, North Carolina, United States of America.
PLoS One. 2022 Oct 25;17(10):e0273746. doi: 10.1371/journal.pone.0273746. eCollection 2022.
The elementary steps of transcription as catalyzed by E. coli RNA polymerase during one and two rounds of the nucleotide addition cycle (NAC) were resolved in rapid kinetic studies. Modelling of stopped-flow kinetic data of pyrophosphate release in a coupled enzyme assay during one round of the NAC indicates that the rate of pyrophosphate release is significantly less than that for nucleotide incorporation. Upon modelling of the stopped-flow kinetic data for pyrophosphate release during two rounds of the NAC, it was observed that the presence of the next nucleotide for incorporation increases the rate of release of the first pyrophosphate equivalent; incorrect nucleotides for incorporation had no effect on the rate of pyrophosphate release. Although the next nucleotide for incorporation increases the rate of pyrophosphate release, it is still significantly less than the rate of incorporation of the first nucleotide. The results from the stopped-flow kinetic studies were confirmed by using quench-flow followed by thin-layer chromatography (QF-TLC) with only the first nucleotide for incorporation labeled on the gamma phosphate with 32P to monitor pyrophosphate release. Collectively, the results are consistent with an NTP-driven model for the NAC in which the binding of the next cognate nucleotide for incorporation causes a synergistic conformational change in the enzyme that triggers the more rapid release of pyrophosphate, translocation of the enzyme along the DNA template strand and nucleotide incorporation.
在快速动力学研究中，解析了大肠杆菌 RNA 聚合酶在核苷酸添加循环 (NAC) 的一轮和两轮中催化转录的基本步骤。对 NAC 一轮中焦磷酸释放的偶联酶测定的停流动力学数据进行建模表明，焦磷酸释放的速率明显低于核苷酸掺入的速率。对 NAC 两轮中焦磷酸释放的停流动力学数据进行建模后，观察到下一个用于掺入的核苷酸的存在增加了第一个焦磷酸当量的释放速率；错误掺入的核苷酸对焦磷酸释放速率没有影响。尽管下一个用于掺入的核苷酸增加了焦磷酸释放的速率，但它仍然明显低于第一个核苷酸掺入的速率。停流动力学研究的结果通过使用仅标记在 γ 磷酸上的第一个掺入核苷酸的 32P 的快速淬火流动 followed by thin-layer chromatography (QF-TLC) 得到了证实，以监测焦磷酸的释放。总的来说，这些结果与 NAC 的 NTP 驱动模型一致，其中下一个用于掺入的互补核苷酸的结合导致酶的协同构象变化，从而引发更快速的焦磷酸释放、酶沿着 DNA 模板链的转位和核苷酸掺入。
