Bridge helix bending promotes RNA polymerase II backtracking through a critical and conserved threonine residue.
Bridge helix bending promotes RNA polymerase II backtracking through a critical and conserved threonine residue.
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桥螺旋弯曲促进 RNA 聚合酶 II 通过关键且保守的苏氨酸残基回溯
DOI：
10.1038/ncomms11244
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发表时间：
2016-04-19
影响因子：
16.6
通讯作者：
Huang X
中科院分区：
文献类型：
--
作者：
Da LT;Pardo-Avila F;Xu L;Silva DA;Zhang L;Gao X;Wang D;Huang X
RNA聚合酶II（Pol II）回溯过程的动力学知之甚少。我们建立了一个马尔可夫状态模型，从广泛的分子动力学模拟，以确定亚稳态的中间状态和动力学回溯原子的细节。我们的研究结果表明，Pol II回溯发生在一个逐步的模式，其中涉及两个中间状态。我们发现桥螺旋（BH）的连续弯曲运动是一个关键的检查点，利用高度保守的BH残基T831作为RNA：DNA杂交体3′端碱基配对的传感探针。如果碱基对错配，BH弯曲可以促进RNA 3′端核苷酸进入磨损状态，进一步导致回溯状态。这些计算观察结果通过定点突变和转录物切割测定得到验证，并提供了对调节向后易位偏好的关键因素的见解。 RNA聚合酶II可以通过需要酶回溯的校对机制检测和切割错误掺入的核苷酸。在这里，作者表明Pol II回溯以逐步模式发生，涉及两个中间状态，其中末端RNA核苷酸的磨损是先决条件。
The dynamics of the RNA polymerase II (Pol II) backtracking process is poorly understood. We built a Markov State Model from extensive molecular dynamics simulations to identify metastable intermediate states and the dynamics of backtracking at atomistic detail. Our results reveal that Pol II backtracking occurs in a stepwise mode where two intermediate states are involved. We find that the continuous bending motion of the Bridge helix (BH) serves as a critical checkpoint, using the highly conserved BH residue T831 as a sensing probe for the 3′-terminal base paring of RNA:DNA hybrid. If the base pair is mismatched, BH bending can promote the RNA 3′-end nucleotide into a frayed state that further leads to the backtracked state. These computational observations are validated by site-directed mutagenesis and transcript cleavage assays, and provide insights into the key factors that regulate the preferences of the backward translocation. RNA Polymerase II can detect and cleave mis-incorporated nucleotides by a proofreading mechanism that requires backtracking of the enzyme. Here the authors show that Pol II backtracking occurs in a stepwise mode that involves two intermediate states where the fraying of the terminal RNA nucleotide is a prerequisite.