Comparing FRET Pairs that Report on Intersubunit Rotation in Bacterial Ribosomes.
EF-G
intersubunit rotation
ribosome
single-molecule FRET
translocation
Journal
Journal of molecular biology
ISSN: 1089-8638
Titre abrégé: J Mol Biol
Pays: Netherlands
ID NLM: 2985088R
Informations de publication
Date de publication:
01 08 2023
01 08 2023
Historique:
received:
01
02
2023
revised:
03
05
2023
accepted:
14
06
2023
pmc-release:
01
08
2024
medline:
21
7
2023
pubmed:
23
6
2023
entrez:
22
6
2023
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 37348753
pii: S0022-2836(23)00283-8
doi: 10.1016/j.jmb.2023.168185
pmc: PMC10528089
mid: NIHMS1911940
pii:
doi:
Substances chimiques
Guanosine Triphosphate
86-01-1
Peptide Elongation Factor G
0
Ribosomal Proteins
0
RNA, Ribosomal, 23S
0
RNA, Transfer
9014-25-9
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
168185Subventions
Organisme : NIGMS NIH HHS
ID : R35 GM141812
Pays : United States
Commentaires et corrections
Type : UpdateOf
Informations de copyright
Copyright © 2023 Elsevier Ltd. All rights reserved.
Déclaration de conflit d'intérêts
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.
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