Conformational sampling of aminoacyl-tRNA during selection on the bacterial ribosome.
Conformational sampling of aminoacyl-tRNA during selection on the bacterial ribosome.
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DOI：
10.1016/j.jmb.2010.04.038
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发表时间：
2010-06-18
影响因子：
5.6
通讯作者：
Blanchard SC
中科院分区：
文献类型：
--
作者：
Geggier P;Dave R;Feldman MB;Terry DS;Altman RB;Munro JB;Blanchard SC
氨酰基-tRNA (aa-tRNA) 与延伸因子-Tu (EF-Tu) 和 GTP 形成三元复合物，通过多步骤、mRNA 密码子依赖性机制进入核糖体的氨酰基 (A) 位点。这个过程会优先选择每个 mRNA 密码子的同源 aa-tRNA，从而提高基因表达的保真度。核糖体通过识别在其解码位点启动的正确底物的结构特征来积极促进这一过程，以加速 EF-Tu 催化的 GTP 水解和核糖体催化的肽键形成的速率。在这里，使用单分子荧光共振能量转移 (smFRET) 从多个结构角度研究了支撑 aa-tRNA 选择过程的构象事件的顺序和时间。这些测量的时间分辨率扩展到 2.5 和 10 毫秒，比之前的研究提高了 10-50 倍。获得的数据表明，aa-tRNA 在 GTP 水解之前和之后在 A 位点内经历快速构象采样。这表明 aa-tRNA 与不可逆 GTP 水解和肽键形成所需的结构元件的比对在保真度机制中起着关键作用。这些观察结果提供了直接证据，表明选择过程受 A 位点内 aa-tRNA 运动的控制，为物理框架增添了新的见解，有助于解释 GTP 水解和肽键形成的速率如何受 mRNA 密码子和系统内其他保真度决定因素的控制。
Aminoacyl-tRNA (aa-tRNA), in a ternary complex with Elongation Factor-Tu (EF-Tu) and GTP, enters the aminoacyl (A) site of the ribosome via a multi-step, mRNA codon-dependent mechanism. This process gives rise to the preferential selection of cognate aa-tRNAs for each mRNA codon and consequently the fidelity of gene expression. The ribosome actively facilitates this process by recognizing structural features of the correct substrate, initiated in its decoding site, to accelerate the rates of EF-Tu-catalyzed GTP hydrolysis and ribosome-catalyzed peptide bond formation. Here, the order and timing of conformational events underpinning the aa-tRNA selection process were investigated from multiple structural perspectives using single-molecule fluorescence resonance energy transfer (smFRET). The time resolution of these measurements was extended to 2.5 and 10ms, a 10–50-fold improvement over previous studies. The data obtained reveal that aa-tRNA undergoes fast conformational sampling within the A site, both before and after GTP hydrolysis. This suggests that the alignment of aa-tRNA with respect to structural elements required for irreversible GTP hydrolysis and peptide bond formation plays a key role in the fidelity mechanism. These observations provide direct evidence that the selection process is governed by motions of aa-tRNA within the A site, adding new insights into the physical framework that helps explain how the rates of GTP hydrolysis and peptide bond formation are controlled by the mRNA codon and other fidelity determinants within the system.
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DOI：
10.1073/pnas.0705988104
发表时间：
2007-08-21
影响因子：
11.1
作者：
Lee, Tae-Hee;Blanchard, Scott C.;Chu, Steven
通讯作者：
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影响因子：
3.4
作者：
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通讯作者：
Blanchard, Scott C.
影响因子：
64.5
作者：
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通讯作者：
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影响因子：
3.4
作者：
McKinney, Sean A.;Joo, Chirlmin;Ha, Taekjip
通讯作者：
Ha, Taekjip
影响因子：
3.9
作者：
NINIO, J
通讯作者：
NINIO, J