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 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.
Cornish
Peter V
PV
Department of Physics, University of Illinois, 1110 West Green Street, Urbana, IL 61801, USA.
Ermolenko
Dmitri N
DN
Noller
Harry F
HF
Ha
Taekjip
T
D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D020653:Peptide Elongation Factor G / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism*; D012399:Rotation; D046249:Transfer RNA Aminoacylation
D020653:Peptide Elongation Factor G; D021122:Protein Subunits; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012343:RNA, Transfer