Step-by-step progress toward understanding the hepatitis C virus RNA helicase.
Step-by-step progress toward understanding the hepatitis C virus RNA helicase.
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DOI：
10.1002/hep.21200
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发表时间：
2006-06-01
期刊：
影响因子：
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通讯作者：
Frick, David N
中科院分区：
文献类型：
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作者：
Frick, David N
解旋酶是一类普遍存在的酶，涉及 DNA 和 RNA 代谢的几乎所有方面。尽管最近在了解其作用机制方面取得了进展，但有限的分辨率使得人们无法了解这些酶将核酸结构重排与 ATP 结合和水解耦合的详细机制。观察这些运动蛋白的个体机械循环对于理解它们的细胞功能至关重要。在这里，我们以两个碱基对和 20 毫秒的分辨率实时跟踪丙型肝炎病毒解旋酶 (NS3) 单体的 RNA 易位和解旋周期。 NS3 是病毒复制所必需的代表性超家族 2 解旋酶，因此是一个潜在的重要药物靶点。我们表明，NS3 的循环运动是由 ATP 以 11 +/- 3 碱基对的离散步骤协调的，并且实际的解旋发生在 3.6 +/- 1.3 碱基对的快速较小子步骤中，这也是由 ATP 结合触发的，表明 NS3 可能像尺蠖一样移动。这种 ATP 偶联机制可能适用于参与许多重要细胞功能的其他非六聚体解旋酶。这里开发的测定应该可用于研究广泛的核酸易位马达。
Helicases are a ubiquitous class of enzymes involved in nearly all aspects of DNA and RNA metabolism. Despite recent progress in understanding their mechanism of action, limited resolution has left inaccessible the detailed mechanisms by which these enzymes couple the rearrangement of nucleic acid structures to the binding and hydrolysis of ATP. Observing individual mechanistic cycles of these motor proteins is central to understanding their cellular functions. Here we follow in real time, at a resolution of two base pairs and 20 ms, the RNA translocation and unwinding cycles of a hepatitis C virus helicase (NS3) monomer. NS3 is a representative superfamily-2 helicase essential for viral replication, and therefore a potentially important drug target. We show that the cyclic movement of NS3 is coordinated by ATP in discrete steps of 11 +/- 3 base pairs, and that actual unwinding occurs in rapid smaller substeps of 3.6 +/- 1.3 base pairs, also triggered by ATP binding, indicating that NS3 might move like an inchworm. This ATP-coupling mechanism is likely to be applicable to other non-hexameric helicases involved in many essential cellular functions. The assay developed here should be useful in investigating a broad range of nucleic acid translocation motors.