Desai Varsha P, Frank Filipp, Lee Antony, Righini Maurizio, Lancaster Laura, Noller Harry F, Tinoco Ignacio, Bustamante Carlos
Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Jason L. Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, Berkeley, CA 94720, USA.
Jason L. Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, Berkeley, CA 94720, USA.
Mol Cell. 2019 Sep 5;75(5):1007-1019.e5. doi: 10.1016/j.molcel.2019.07.024. Epub 2019 Aug 27.
The movement of ribosomes on mRNA is often interrupted by secondary structures that present mechanical barriers and play a central role in translation regulation. We investigate how ribosomes couple their internal conformational changes with the activity of translocation factor EF-G to unwind mRNA secondary structures using high-resolution optical tweezers with single-molecule fluorescence capability. We find that hairpin opening occurs during EF-G-catalyzed translocation and is driven by the forward rotation of the small subunit head. Modulating the magnitude of the hairpin barrier by force shows that ribosomes respond to strong barriers by shifting their operation to an alternative 7-fold-slower kinetic pathway prior to translocation. Shifting into a slow gear results from an allosteric switch in the ribosome that may allow it to exploit thermal fluctuations to overcome mechanical barriers. Finally, we observe that ribosomes occasionally open the hairpin in two successive sub-codon steps, revealing a previously unobserved translocation intermediate.
核糖体在信使核糖核酸（mRNA）上的移动常常会被二级结构打断，这些二级结构构成了机械屏障，并在翻译调控中发挥核心作用。我们使用具有单分子荧光能力的高分辨率光镊，研究核糖体如何将其内部构象变化与转位因子EF-G的活性相结合，以解开mRNA二级结构。我们发现，发夹结构的打开发生在EF-G催化的转位过程中，并且由小亚基头部的向前旋转驱动。通过施加力来调节发夹结构的屏障强度，结果表明核糖体在转位之前会通过将其运作转移到另一条速度慢7倍的动力学途径来应对强大的屏障。进入慢速模式是核糖体中一种变构转换的结果，这种变构转换可能使核糖体利用热涨落来克服机械屏障。最后，我们观察到核糖体偶尔会以两个连续的亚密码子步骤打开发夹结构，揭示了一种以前未被观察到的转位中间体。
