Ribosome-induced tuning of GTP hydrolysis by a translational GTPase
Ribosome-induced tuning of GTP hydrolysis by a translational GTPase
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DOI：
10.1073/pnas.1412676111
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发表时间：
2014-10-07
影响因子：
11.1
通讯作者：
Rodnina, Marina V.
中科院分区：
文献类型：
--
作者：
Maracci, Cristina;Peske, Frank;Rodnina, Marina V.
延伸因子Tu （EF-Tu）水解GTP，这是一种将氨基酰基trna传递到核糖体的翻译GTP酶，在解码和翻译保真度中起着关键作用。基本的反应机制和核糖体促进催化的方式是一个有争议的问题。在这里，我们使用突变分析，结合速率/pH谱测量，动力学溶剂同位素效应，以及EF-Tu和核糖体对GTP水解的离子依赖性来剖析反应机理。我们的数据表明，与目前的模型相反，游离EF-Tu中的反应遵循一个不涉及开关II区关键残基H84的途径。在a位点与没有同源密码子的核糖体结合对GTPase的机制影响不大。相比之下，在同源密码子识别后，核糖体诱导EF-Tu重排，使GTP水解对Asp21和His84突变敏感，对K+离子不敏感。我们认为，Asp21和His84提供了一个相互作用的网络，分别稳定了γ -磷酸和亲核水的位置，从而在GTPase作用于核糖体的机制中起间接催化作用。
GTP hydrolysis by elongation factor Tu (EF-Tu), a translational GTPase that delivers aminoacyl-tRNAs to the ribosome, plays a crucial role in decoding and translational fidelity. The basic reaction mechanism and the way the ribosome contributes to catalysis are a matter of debate. Here we use mutational analysis in combination with measurements of rate/pH profiles, kinetic solvent isotope effects, and ion dependence of GTP hydrolysis by EF-Tu off and on the ribosome to dissect the reaction mechanism. Our data suggest that-contrary to current models-the reaction in free EF-Tu follows a pathway that does not involve the critical residue H84 in the switch II region. Binding to the ribosome without a cognate codon in the A site has little effect on the GTPase mechanism. In contrast, upon cognate codon recognition, the ribosome induces a rearrangement of EF-Tu that renders GTP hydrolysis sensitive to mutations of Asp21 and His84 and insensitive to K+ ions. We suggest that Asp21 and His84 provide a network of interactions that stabilize the positions of the gamma-phosphate and the nucleophilic water, respectively, and thus play an indirect catalytic role in the GTPase mechanism on the ribosome.