Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University, New York, NY, USA.
Proc Natl Acad Sci U S A. 2010 Jan 12;107(2):709-14. doi: 10.1073/pnas.0908597107. Epub 2009 Dec 17.
The mechanism of substrate translocation through the ribosome is central to the rapid and faithful translation of mRNA into proteins. The rate-limiting step in translocation is an unlocking process that includes the formation of an "unlocked" intermediate state, which requires the convergence of large-scale conformational events within the ribosome including tRNA hybrid states formation, closure of the ribosomal L1 stalk domain, and subunit ratcheting. Here, by imaging of the pretranslocation ribosome complex from multiple structural perspectives using two- and three-color single-molecule fluorescence resonance energy transfer, we observe that tRNA hybrid states formation and L1 stalk closure, events central to the unlocking mechanism, are not tightly coupled. These findings reveal that the unlocked state is achieved through a stochastic-multistep process, where the extent of conformational coupling depends on the nature of tRNA substrates. These data suggest that cellular mechanisms affecting the coupling of conformational processes on the ribosome may regulate the process of translation elongation.
核糖体中底物转位的机制是 mRNA 快速准确翻译成蛋白质的关键。转位的限速步骤是一个解锁过程，包括形成“解锁”的中间状态，这需要核糖体内的大规模构象事件的收敛，包括 tRNA 杂交状态的形成、核糖体 L1 茎结构域的闭合以及亚基棘轮运动。在这里，我们通过使用双色和三色单分子荧光共振能量转移从多个结构角度对前转位核糖体复合物进行成像，观察到 tRNA 杂交状态的形成和 L1 茎的闭合，这些是解锁机制的核心事件，它们之间没有紧密的偶联。这些发现表明，解锁状态是通过随机多步过程实现的，其中构象偶联的程度取决于 tRNA 底物的性质。这些数据表明，影响核糖体上构象过程偶联的细胞机制可能调节翻译延伸过程。
