Detection of sequential polyubiquitylation on a millisecond timescale.
Detection of sequential polyubiquitylation on a millisecond timescale.
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泛素链通过由E1， E2和E3组成的级联酶在底物上产生的途径尚不清楚。已经提出了多个不同的模型，包括在E2或底物上的链组装。然而，反应的速度和复杂性阻碍了直接的实验测试来区分潜在的途径。在这里，我们引入新的理论和实验方法来解决这两个限制。基于产物分布的定量框架预测，真正有趣的新基因（RING） E3s SCFCdc4和SCFβ-TrCP通过单个泛素的顺序转移与E2 Cdc34一起在底物上构建多泛素链。测量与毫秒时间分辨率直接证明了底物多泛素化顺序进行。我们的研究结果为环泛素连接酶的机制提供了前所未有的一瞥，并阐明了泛素链组装速率和模式的定量参数。
The pathway by which ubiquitin chains are generated on substrate via a cascade of enzymes consisting of an E1, E2 and E3 remains unclear. Multiple distinct models involving chain assembly on E2 or substrate have been proposed. However, the speed and complexity of the reaction have precluded direct experimental tests to distinguish between potential pathways. Here we introduce new theoretical and experimental methodologies to address both limitations. A quantitative framework based on product distribution predicts that the really interesting new gene (RING) E3s SCFCdc4 and SCFβ-TrCP work with the E2 Cdc34 to build polyubiquitin chains on substrates by sequential transfers of single ubiquitins. Measurements with millisecond time resolution directly demonstrate that substrate polyubiquitylation proceeds sequentially. Our results present an unprecedented glimpse into the mechanism of RING ubiquitin ligases and illuminate the quantitative parameters that underlie the rate and pattern of ubiquitin chain assembly.
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