Initial binding of the elongation factor Tu center dot GTP center dot aminoacyl-tRNA complex preceding codon recognition on the ribosome
Initial binding of the elongation factor Tu center dot GTP center dot aminoacyl-tRNA complex preceding codon recognition on the ribosome
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DOI：
10.1074/jbc.271.2.646
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发表时间：
1996-01-12
影响因子：
4.8
通讯作者：
Wintermeyer, W
中科院分区：
文献类型：
--
作者：
Rodnina, MV;Pape, T;Wintermeyer, W
在核糖体与延伸因子Tu（EF - Tu）、GTP和氨酰 - tRNA复合物之间相互作用的序列中，最终导致氨酰 - tRNA结合在A位点的第一步是一个不依赖密码子的初始复合物的形成。我们使用几种荧光tRNA衍生物，通过时间分辨（停流）和稳态荧光测量对初始结合以及所形成的复合物进行了表征。该复合物不稳定，其结合和解离的速率常数分别为6×10⁷ M⁻¹ s⁻¹和24 s⁻¹（20℃，10 mM Mg²⁺）。我们确定了初始结合的热力学和活化参数，并估计有5个Mg²⁺离子参与相互作用。虽然同源三元复合物从初始结合经过密码子识别到快速的GTP水解，但非同源复合物中GTP水解的速率常数要低4个数量级，尽管在两种情况下初始复合物都能快速形成。因此，EF - Tu的核糖体诱导的GTP水解受到tRNA存在的强烈影响。这表明在初始结合复合物形成之后发生的密码子 - 反密码子识别提供了一个特定信号，触发EF - Tu在核糖体上快速水解GTP。
The first step in the sequence of interactions between the ribosome and the complex of elongation factor Tu (EF-Tu), GTP, and aminoacyl-tRNA, which eventually leads to A site-bound aminoacyl-tRNA, is the codon-independent formation of an initial complex. We have characterized the initial binding and the resulting complex by time resolved (stopped-flow) and steady-state fluorescence measurements using several fluorescent tRNA derivatives. The complex is labile, with rate constants of 6 x 10(7) M(-1) s(-1) and 24 s(-1) (20 degrees C, 10 mM Mg2+) for binding and dissociation, respectively. Both thermodynamic and activation parameters of initial binding were determined, and five Mg2+ ions were estimated to participate in the interaction. While a cognate ternary complex proceeds from initial binding through codon recognition to rapid GTP hydrolysis, the rate constant of GTP hydrolysis in the non-cognate complex is 4 orders of magnitude lower, despite the rapid formation of the initial complex in both cases, Hence, the ribosome-induced GTP hydrolysis by EF-Tu is strongly affected by the presence of the tRNA. This suggests that codon-anticodon recognition, which takes place after the formation of the initial binding complex, provides a specific signal that triggers fast GTP hydrolysis by EF-Tu on the ribosome.