Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites.
The elongation cycle of protein synthesis involves the delivery of aminoacyl-transfer RNAs to the aminoacyl-tRNA-binding site (A site) of the ribosome, followed by peptide-bond formation and translocation of the tRNAs through the ribosome to reopen the A site. The translocation reaction is catalysed by elongation factor G (EF-G) in a GTP-dependent manner. Despite the availability of structures of various EF-G-ribosome complexes, the precise mechanism by which tRNAs move through the ribosome still remains unclear. Here we use multiparticle cryoelectron microscopy analysis to resolve two previously unseen subpopulations within Thermus thermophilus EF-G-ribosome complexes at subnanometre resolution, one of them with a partly translocated tRNA. Comparison of these substates reveals that translocation of tRNA on the 30S subunit parallels the swivelling of the 30S head and is coupled to unratcheting of the 30S body. Because the tRNA maintains contact with the peptidyl-tRNA-binding site (P site) on the 30S head and simultaneously establishes interaction with the exit site (E site) on the 30S platform, a novel intra-subunit 'pe/E' hybrid state is formed. This state is stabilized by domain IV of EF-G, which interacts with the swivelled 30S-head conformation. These findings provide direct structural and mechanistic insight into the 'missing link' in terms of tRNA intermediates involved in the universally conserved translocation process.
Institut für Medizinische Physik und Biophysik, Charite - Universitätsmedizin Berlin, Ziegelstrasse 5-9, 10117-Berlin, Germany.
Andreas H
AH
Ratje
Justus
J
Loerke
Aleksandra
A
Mikolajka
Matthias
M
Brünner
Peter W
PW
Hildebrand
Agata L
AL
Starosta
Alexandra
A
Dönhöfer
Sean R
SR
Connell
Paola
P
Fucini
Thorsten
T
Mielke
Paul C
PC
Whitford
José N
JN
Onuchic
Yanan
Y
Yu
Karissa Y
KY
Sanbonmatsu
Roland K
RK
Hartmann
Pawel A
PA
Penczek
Daniel N
DN
Wilson
Christian M T
CM
Spahn
D001665:Binding Sites; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009068:Movement; D020653:Peptide Elongation Factor G / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D054680:Ribosome Subunits, Small, Bacterial / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D016963:Thermus thermophilus / Q000737:chemistry
D020653:Peptide Elongation Factor G; D021122:Protein Subunits; D006153:Guanosine Diphosphate; D012343:RNA, Transfer