The ATP-dependent remodeler RSC transfers histone dimers and octamers through the rapid formation of an unstable encounter intermediate.
The ATP-dependent remodeler RSC transfers histone dimers and octamers through the rapid formation of an unstable encounter intermediate.
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DOI：
10.1021/bi101491u
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发表时间：
2010-11-16
期刊：
影响因子：
2.9
通讯作者：
Narlikar, Geeta J.
中科院分区：
文献类型：
--
作者：
Rowe, Claire E.;Narlikar, Geeta J.
RSC是出芽酵母中一种重要的染色质重塑复合体，参与多种生物过程，包括转录、重组、修复和复制。RSC是如何参与这些不同的过程的，目前还不完全清楚。在体外，RSC利用ATP进行几个看似不同的反应：它重新定位核小体，在核小体之间转移H2A/H2B二聚体，在DNA片段之间转移组蛋白八聚体。这就提出了一个有趣的机制问题，即这种分子机器是如何使用单个atp酶亚基来产生这些不同的产物的。在这里，我们使用基于fret的方法对RSC反应的产物进行动力学排序。令人惊讶的是，当用FRET测定时，H2A/H2B二聚体和组蛋白八聚体的转移在几秒的时间尺度上开始，但当用天然凝胶测定时，稳定的核小体产物的形成在几分钟的时间尺度上发生。这些结果表明一个模型，其中RSC作用迅速产生一个不稳定的相遇中间体，其中包含两个交换底物在近距离。然后，这种中间体更慢地坍塌，形成在天然凝胶上看到的稳定的转移产物。产生转移产物的快速、生物学相关的时间尺度意味着这些产物在体内可以发挥关键作用。
RSC, an essential chromatin remodeling complex in budding yeast, is involved in a variety of biological processes including transcription, recombination, repair and replication. How RSC participates in such diverse processes is not fully understood. In vitro, RSC uses ATP to carry out several seemingly distinct reactions: it repositions nucleosomes, transfers H2A/H2B dimers between nucleosomes and transfers histone octamers between pieces of DNA. This raises the intriguing mechanistic question of how this molecular machine can use a single ATPase subunit to create these varied products. Here, we use a FRET-based approach to kinetically order the products of the RSC reaction. Surprisingly, transfer of H2A/H2B dimers and histone octamers is initiated on a time scale of seconds when assayed by FRET, but formation of stable nucleosomal products occurs on a time scale of minutes when assayed by native gel. These results suggest a model in which RSC action rapidly generates an unstable encounter intermediate that contains the two exchange substrates in close proximity. This intermediate then collapses more slowly to form the stable transfer products seen on native gels. The rapid, biologically relevant time scale on which the transfer products are generated implies that such products can play key roles in vivo.
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