Molecular dynamics studies of the energetics of translocation in model T7 RNA polymerase elongation complexes.
Molecular dynamics studies of the energetics of translocation in model T7 RNA polymerase elongation complexes.
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DOI：
10.1002/prot.22134
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发表时间：
2008-12
影响因子：
2.9
通讯作者：
Sousa, Rui
中科院分区：
文献类型：
--
作者：
Woo, Hyung-June;Liu, Yuemin;Sousa, Rui
通过分子动力学模拟研究了单亚基 T7 RNA 聚合酶延伸复合物中的易位，使用指结构域开放的转位后晶体结构（在没有焦磷酸盐、镁离子和核苷酸底物的情况下稳定的中间体）。进行无约束和伞式采样模拟来检查易位的能量学。使用代表RNA-DNA杂交碱基对相对于参考结构的平均和个体位移的反应坐标来量化易位的程度。此外，还对手指结构域关闭但去除了焦磷酸盐和镁的产物复合物进行了无约束模拟，以检查焦磷酸盐释放后预转位关闭状态的局部稳定性。研究发现，在开放状态下，整个混合体的平均空间运动在易位后到易位前方向上的能量消耗很大，而易位前状态在封闭复合体中是稳定的，这支持了构象状态决定易位状态的全局稳定性的观点。然而，开放构象中RNA 3′端的空间波动很大，典型范围达到3-4 Å。我们的结果表明，热波动在单个核苷酸的易位中比在大段核苷酸链的运动中发挥更重要的作用：RNA 3'端可以在单一构象状态下移入和移出活性位点，而如果没有构象变化，杂交体的整体运动可能在热力学上是不利的。
Translocation in the single subunit T7 RNA polymerase elongation complex was studied by molecular dynamics simulations using the posttrans-located crystal structure with the fingers domain open, an intermediate stable in the absence of pyrophosphate, magnesium ions, and nucleotide substrate. Unconstrained and umbrella sampling simulations were performed to examine the energetics of translocations. The extent of translocation was quantified using reaction coordinates representing the average and individual displacements of the RNA-DNA hybrid base pairs with respect to a reference structure. In addition, an unconstrained simulation was also performed for the product complex with the fingers domain closed, but with the pyrophosphate and magnesium removed, in order to examine the local stability of the pretranslocated closed state after the pyrophosphate release. The average spatial movement of the entire hybrid was found to be energetically costly in the post- to pretranslocated direction in the open state, while the pretranslocated state was stable in the closed complex, supporting the notion that the conformational state dictates the global stability of translocation states. However, spatial fluctuations of the RNA 3′-end in the open conformation were extensive, with the typical range reaching 3–4 Å. Our results suggest that thermal fluctuations play more important roles in the translocation of individual nucleotides than in the movement of large sections of nucleotide strands: RNA 3′-end can move into and out of the active site within a single conformational state, while a global movement of the hybrid may be thermodynamically unfavorable without the conformational change.
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影响因子：
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作者：
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