The tRNA-modifying function of MnmE is controlled by post-hydrolysis steps of its GTPase cycle.
The tRNA-modifying function of MnmE is controlled by post-hydrolysis steps of its GTPase cycle.
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DOI：
10.1093/nar/gkt320
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发表时间：
2013-07
影响因子：
14.9
通讯作者：
Armengod ME
中科院分区：
文献类型：
--
作者：
Prado S;Villarroya M;Medina M;Armengod ME
MnmE是一种参与tRNA修饰的同源二聚体多结构域GTdR。这种蛋白质与Ras样GTP酶的不同之处在于其对鸟嘌呤核苷酸的低亲和力和激活机制，其激活机制是通过其G结构域的顺式、核苷酸和钾依赖性二聚化发生的。此外，MnmE需要GTP水解才具有功能活性。然而，GTP水解如何驱动tRNA修饰以及MnmE GTdR循环如何调节仍然没有解决。在这里，使用停止流和猝灭流技术在单转换条件下研究了MnmE GTPase循环的动力学。我们发现G-结构域解离是整个反应的限速步骤。突变分析和快速动力学分析表明，GTP水解，G-结构域解离和Pi释放可以解偶联，G-结构域解离是直接负责的MnmE的“ON”状态。因此，MnmE提供了一个新的范例，如何打开/关闭循环的GTP酶可以调节细胞过程。我们还表明，MnmE GTdR循环是负控制的反应产物GDP和Pi。这种反馈机制可以防止体内无效的GTP水解。我们提出了一个生物模型，其中tRNA结合所引发的构象变化是必需的，以消除产品的抑制，并启动一个新的GTdR/tRNA修饰周期。
MnmE is a homodimeric multi-domain GTPase involved in tRNA modification. This protein differs from Ras-like GTPases in its low affinity for guanine nucleotides and mechanism of activation, which occurs by a cis, nucleotide- and potassium-dependent dimerization of its G-domains. Moreover, MnmE requires GTP hydrolysis to be functionally active. However, how GTP hydrolysis drives tRNA modification and how the MnmE GTPase cycle is regulated remains unresolved. Here, the kinetics of the MnmE GTPase cycle was studied under single-turnover conditions using stopped- and quench-flow techniques. We found that the G-domain dissociation is the rate-limiting step of the overall reaction. Mutational analysis and fast kinetics assays revealed that GTP hydrolysis, G-domain dissociation and Pi release can be uncoupled and that G-domain dissociation is directly responsible for the ‘ON’ state of MnmE. Thus, MnmE provides a new paradigm of how the ON/OFF cycling of GTPases may regulate a cellular process. We also demonstrate that the MnmE GTPase cycle is negatively controlled by the reaction products GDP and Pi. This feedback mechanism may prevent inefficacious GTP hydrolysis in vivo. We propose a biological model whereby a conformational change triggered by tRNA binding is required to remove product inhibition and initiate a new GTPase/tRNA-modification cycle.
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