Fei Jingyi, Kosuri Pallav, MacDougall Daniel D, Gonzalez Ruben L
Department of Chemistry, Columbia University, New York, NY 10027, USA.
Mol Cell. 2008 May 9;30(3):348-59. doi: 10.1016/j.molcel.2008.03.012.
By using single-molecule fluorescence resonance energy transfer (smFRET), we observe the real-time dynamic coupling between the ribosome, labeled at the L1 stalk, and transfer RNA (tRNA). We find that an interaction between the ribosomal L1 stalk and the newly deacylated tRNA is established spontaneously upon peptide bond formation; this event involves coupled movements of the L1 stalk and tRNAs as well as ratcheting of the ribosome. In the absence of elongation factor G, the entire pretranslocation ribosome fluctuates between just two states: a nonratcheted state, with tRNAs in their classical configuration and no L1 stalk-tRNA interaction, and a ratcheted state, with tRNAs in an intermediate hybrid configuration and a direct L1 stalk-tRNA interaction. We demonstrate that binding of EF-G shifts the equilibrium toward the ratcheted state. Real-time smFRET experiments reveal that the L1 stalk-tRNA interaction persists throughout the translocation reaction, suggesting that the L1 stalk acts to direct tRNA movements during translocation.
通过使用单分子荧光共振能量转移（smFRET）技术，我们观察到标记在L1柄上的核糖体与转运RNA（tRNA）之间的实时动态偶联。我们发现，在肽键形成时，核糖体L1柄与新脱酰基的tRNA之间会自发建立相互作用；这一事件涉及L1柄和tRNA的耦合运动以及核糖体的棘轮运动。在没有延伸因子G的情况下，整个转位前核糖体仅在两种状态之间波动：一种是非棘轮状态，tRNA处于经典构象且不存在L1柄-tRNA相互作用；另一种是棘轮状态，tRNA处于中间杂交构象且存在直接的L1柄-tRNA相互作用。我们证明，延伸因子G的结合会使平衡向棘轮状态移动。实时smFRET实验表明，L1柄-tRNA相互作用在整个转位反应过程中持续存在，这表明L1柄在转位过程中起到引导tRNA运动的作用。
