tRNA dynamics on the ribosome during translation
tRNA dynamics on the ribosome during translation
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DOI：
10.1073/pnas.0403884101
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发表时间：
2004-08-31
影响因子：
11.1
通讯作者：
Chu, S
中科院分区：
文献类型：
--
作者：
Blanchard, SC;Kim, HD;Chu, S
利用单分子荧光光谱，在表面固定的核糖体中，荧光标记的tRNA之间的时间分辨构象变化经历了一个完整的翻译延伸周期。荧光共振能量转移观察了氨基酰基trna （aa-tRNA）通过多步、延伸因子- tu依赖的过程稳定地进入核糖体的氨基酰基位点（A位点）。随后，结合在肽基位点和A位点的tRNA分子在经典态和杂化态两种构型之间波动。在A位点携带aa-tRNA和肽基-tRNA的配合物的经典态和杂化态寿命测量表明，肽键的形成使经典态tRNA构型的寿命减少了大约6倍。这些数据表明，生长的肽链在调节杂交和经典状态之间的波动中起作用。单分子荧光共振能量转移也用于观察aa-tRNA调节与延伸因子g介导的易位。tRNA构型的动态重排也在易位反应后被观察到。这项工作强调了核糖体功能动力学的重要性，并在两个以上组分的系统中展示了单颗粒酶学。
Using single-molecule fluorescence spectroscopy, time-resolved conformational changes between fluorescently labeled tRNA have been characterized within surface-immobilized ribosomes proceeding through a complete cycle of translation elongation. Fluorescence resonance energy transfer was used to observe aminoacyl-tRNA (aa-tRNA) stably accommodating into the aminoacyl site (A site) of the ribosome via a multistep, elongation factor-Tu dependent process. Subsequently, tRNA molecules, bound at the peptidyl site and A site, fluctuate between two configurations assigned as classical and hybrid states. The lifetime of classical and hybrid states, measured for complexes carrying aa-tRNA and peptidyl-tRNA at the A site, shows that peptide bond formation decreases the lifetime of the classical-state tRNA configuration by approximate to6-fold. These data suggest that the growing peptide chain plays a role in modulating fluctuations between hybrid and classical states. Single-molecule fluorescence resonance energy transfer was also used to observe aa-tRNA accommodation coupled with elongation factor G-mediated translocation. Dynamic rearrangements in tRNA configuration are also observed subsequent to the translocation reaction. This work underscores the importance of dynamics in ribosome function and demonstrates single-particle enzymology in a system of more than two components.