Spontaneous Intersubunit Rotation in Single Ribosomes

During the elongation cycle, tRNA and mRNA undergo coupled translocation through the ribosome catalyzed by elongation factor G (EF-G). Cryo-EM reconstructions of certain EF-G-containing complexes led to the proposal that the mechanism of translocation involves rotational movement between the two rib...

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Bibliographic Details
Published in:Molecular cell 2008-06, Vol.30 (5), p.578-588
Main Authors: Cornish, Peter V., Ermolenko, Dmitri N., Noller, Harry F., Ha, Taekjip
Format: Article
Language:English
Subjects:
Escherichia coli - enzymology
Escherichia coli - genetics
Fluorescence Resonance Energy Transfer
Kinetics
Peptide Elongation Factor G - metabolism
Protein Binding
Protein Biosynthesis
Protein Subunits - chemistry
Protein Subunits - metabolism
Protein Transport
Ribosomes - chemistry
Ribosomes - metabolism
RNA
RNA, Bacterial - genetics
RNA, Bacterial - metabolism
RNA, Messenger - metabolism
RNA, Transfer - genetics
RNA, Transfer - metabolism
Rotation
Transfer RNA Aminoacylation
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Summary:During the elongation cycle, tRNA and mRNA undergo coupled translocation through the ribosome catalyzed by elongation factor G (EF-G). Cryo-EM reconstructions of certain EF-G-containing complexes led to the proposal that the mechanism of translocation involves rotational movement between the two ribosomal subunits. Here, using single-molecule FRET, we observe that pretranslocation ribosomes undergo spontaneous intersubunit rotational movement in the absence of EF-G, fluctuating between two conformations corresponding to the classical and hybrid states of the translocational cycle. In contrast, posttranslocation ribosomes are fixed predominantly in the classical, nonrotated state. Movement of the acceptor stem of deacylated tRNA into the 50S E site and EF-G binding to the ribosome both contribute to stabilization of the rotated, hybrid state. Furthermore, the acylation state of P site tRNA has a dramatic effect on the frequency of intersubunit rotation. Our results provide direct evidence that the intersubunit rotation that underlies ribosomal translocation is thermally driven.
ISSN:1097-2765
1097-4164
DOI:10.1016/j.molcel.2008.05.004