Transient reversal of RNA polymerase II active site closing controls fidelity of transcription elongation
Transient reversal of RNA polymerase II active site closing controls fidelity of transcription elongation
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DOI：
10.1016/j.molcel.2008.04.017
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发表时间：
2008-06-06
期刊：
影响因子：
16
通讯作者：
Kashlev, Mikhail
中科院分区：
文献类型：
--
作者：
Kireeva, Maria L.;Nedialkov, Yuri A.;Kashlev, Mikhail
为了研究RNA聚合酶II（POL II）的保真度，我们分析了RBPL（RPO21）的6-唑他服敏感性和TFIIS依赖性E1103G突变体的性质，RBPL（RPO21）是编码Cervisiae中POL II的催化亚基的基因。使用基于体内重新转录的转录保真度测定法，我们观察到RPB1-E1103G导致转录误差增加了3倍。该突变体在体外显示转录伸长的忠诚度降低了10倍。在停滞的伸长络合物中，该突变似乎没有显着影响POL II的易位状态平衡。主要是，它促进了聚合酶活性中心中的NTP隔离。此外，稳态的状态分析表明，E1103G突变将封闭和开放的活动中心构象之间的平衡转移到封闭形式。因此，活跃中心的开放构象是对结构前保真控制必不可少的中间构象。相似的机制可以控制DNA依赖性DNA聚合酶和RNA依赖性RNA聚合酶的保真度。
To study fidelity of RNA polymerase II (Pol II), we analyzed properties of the 6-azauracil-sensitive and TFIIS-dependent E1103G mutant of rbpl (rpo21), the gene encoding the catalytic subunit of Pol II in Saccharomyces cerevisiae. Using an in vivo retro-transposition-based transcription fidelity assay, we observed that rpb1-E1103G causes a 3-fold increase in transcription errors. This mutant showed a 10-fold decrease in fidelity of transcription elongation in vitro. The mutation does not appear to significantly affect translocation state equilibrium of Pol II in a stalled elongation complex. Primarily, it promotes NTP sequestration in the polymerase active center. Furthermore, pre-steady-state analyses revealed that the E1103G mutation shifted the equilibrium between the closed and the open active center conformations toward the closed form. Thus, open conformation of the active center emerges as an intermediate essential for preincorporation fidelity control. Similar mechanisms may control fidelity of DNA-dependent DNA polymerases and RNA-dependent RNA polymerases.