Comparing FRET Pairs that Report on Intersubunit Rotation in Bacterial Ribosomes.

Autor: Das A; Department of Biochemistry & Biophysics, School of Medicine and Dentistry, and Center for RNA Biology, University of Rochester, Rochester, NY 14642, United States., Bao C; Department of Biochemistry & Biophysics, School of Medicine and Dentistry, and Center for RNA Biology, University of Rochester, Rochester, NY 14642, United States., Ermolenko DN; Department of Biochemistry & Biophysics, School of Medicine and Dentistry, and Center for RNA Biology, University of Rochester, Rochester, NY 14642, United States. Electronic address: Dmitri_Ermolenko@URMC.Rochester.edu.
Jazyk: angličtina
Zdroj: Journal of molecular biology [J Mol Biol] 2023 Aug 01; Vol. 435 (15), pp. 168185. Date of Electronic Publication: 2023 Jun 20.
DOI: 10.1016/j.jmb.2023.168185
Abstrakt: 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.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2023 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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