Comparing FRET Pairs that Report on Intersubunit Rotation in Bacterial Ribosomes

Mediated by elongation factor G (EF-G), ribosome translocation along mRNA is accompanied by rota-tional 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 intersub-unit 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 sponta-neously 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. & COPY; 2023 Elsevier Ltd. All rights reserved.

Automated summary

In this study, two independent FRET assays were used to confirm that pre-translocation ribosomes without EF-G can undergo spontaneous intersubunit rotations between non-rotated and rotated conformations. The fraction of spontaneously fluctuating molecules changes depending on experimental conditions, highlighting the importance of using multiple FRET assays in studies of ribosome dynamics and emphasizing the role of thermally-driven large-scale ribosome rearrangements in translation.