Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University, New York, NY 10021, USA.
EMBO J. 2010 Feb 17;29(4):770-81. doi: 10.1038/emboj.2009.384. Epub 2009 Dec 24.
A key intermediate in translocation is an 'unlocked state' of the pre-translocation ribosome in which the P-site tRNA adopts the P/E hybrid state, the L1 stalk domain closes and ribosomal subunits adopt a ratcheted configuration. Here, through two- and three-colour smFRET imaging from multiple structural perspectives, EF-G is shown to accelerate structural and kinetic pathways in the ribosome, leading to this transition. The EF-G-bound ribosome remains highly dynamic in nature, wherein, the unlocked state is transiently and reversibly formed. The P/E hybrid state is energetically favoured, but exchange with the classical P/P configuration persists; the L1 stalk adopts a fast dynamic mode characterized by rapid cycles of closure and opening. These data support a model in which P/E hybrid state formation, L1 stalk closure and subunit ratcheting are loosely coupled, independent processes that must converge to achieve the unlocked state. The highly dynamic nature of these motions, and their sensitivity to conformational and compositional changes in the ribosome, suggests that regulating the formation of this intermediate may present an effective avenue for translational control.
易位的关键中间态是前易位核糖体的“解锁状态”，在此状态下 P 位 tRNA 采用 P/E 杂交态，L1 茎部结构域关闭，核糖体亚基呈棘轮式构型。在此，通过从多个结构角度的双色和三色 smFRET 成像，显示 EF-G 可加速核糖体中的结构和动力学途径，从而导致这种转变。EF-G 结合的核糖体在本质上仍保持高度动态，其中解锁状态是短暂且可逆形成的。P/E 杂交态在能量上是有利的，但与经典的 P/P 构型的交换仍然存在；L1 茎部呈现快速动态模式，特征是快速的关闭和打开循环。这些数据支持这样一种模型，即 P/E 杂交态的形成、L1 茎部的关闭和亚基的棘轮作用是松散偶联的、独立的过程，必须收敛以达到解锁状态。这些运动的高度动态特性及其对核糖体构象和组成变化的敏感性表明，调节这种中间状态的形成可能是翻译控制的有效途径。
