Das Ananya, Bao Chen, Ermolenko Dmitri N
Department of Biochemistry & Biophysics, School of Medicine and Dentistry, and Center for RNA Biology, University of Rochester, Rochester, NY 14642.
bioRxiv. 2023 May 9:2023.05.09.540051. doi: 10.1101/2023.05.09.540051.
Mediated by elongation factor G (EF-G), ribosome translocation along mRNA is accompanied by rotational movement between ribosomal subunits. Here, we reassess whether the intersubunit rotation requires GTP hydrolysis by EF-G or can occur spontaneously. To that end, we employ two independent FRET assays, which are based on labeling either ribosomal proteins (bS6 and bL9) or rRNAs (h44 of 16S and H101 of 23S rRNA). Both FRET pairs reveal three FRET states, corresponding to the non-rotated, rotated and semi-rotated conformations of the ribosome. Both FRET assays show that in the absence of EF-G, pre-translocation ribosomes containing deacylated P-site tRNA undergo spontaneous intersubunit rotations between non-rotated and rotated conformations. While the two FRET pairs exhibit largely similar behavior, they substantially differ in the fraction of ribosomes showing spontaneous fluctuations. Nevertheless, instead of being an invariable intrinsic property of each FRET pair, the fraction of spontaneously fluctuating molecules changes in both FRET assays depending on experimental conditions. Our results underscore importance of using multiple FRET pairs in studies of ribosome dynamics and highlight the role of thermally-driven large-scale ribosome rearrangements in translation.
在延伸因子G（EF-G）的介导下，核糖体沿mRNA的转位伴随着核糖体亚基之间的旋转运动。在这里，我们重新评估亚基间的旋转是否需要EF-G水解GTP，或者是否可以自发发生。为此，我们采用了两种独立的荧光共振能量转移（FRET）测定法，它们分别基于对核糖体蛋白（bS6和bL9）或核糖体RNA（16S的h44和23S rRNA的H101）进行标记。这两种FRET对都揭示了三种FRET状态，分别对应于核糖体的非旋转、旋转和半旋转构象。两种FRET测定法均表明，在没有EF-G的情况下，含有脱酰化P位点tRNA的转位前核糖体在非旋转和旋转构象之间会自发进行亚基间旋转。虽然这两种FRET对表现出大致相似的行为，但它们在显示自发波动的核糖体比例上有很大差异。然而，自发波动分子的比例并非每种FRET对的不变固有属性，在两种FRET测定法中，其都会根据实验条件而变化。我们的结果强调了在核糖体动力学研究中使用多种FRET对的重要性，并突出了热驱动的大规模核糖体重排在翻译中的作用。
