title: Visualization of two transfer RNAs trapped in transit during elongation factor G-mediated translocation.
abstract: During protein synthesis, coupled translocation of messenger RNAs (mRNA) and transfer RNAs (tRNA) through the ribosome takes place following formation of each peptide bond. The reaction is facilitated by large-scale conformational changes within the ribosomal complex and catalyzed by elongtion factor G (EF-G). Previous structural analysis of the interaction of EF-G with the ribosome used either model complexes containing no tRNA or only a single tRNA, or complexes where EF-G was directly bound to ribosomes in the posttranslocational state. Here, we present a multiparticle cryo-EM reconstruction of a translocation intermediate containing two tRNAs trapped in transit, bound in chimeric intrasubunit ap/P and pe/E hybrid states. The downstream ap/P-tRNA is contacted by domain IV of EF-G and P-site elements within the 30S subunit body, whereas the upstream pe/E-tRNA maintains tight interactions with P-site elements of the swiveled 30S head. Remarkably, a tight compaction of the tRNA pair can be seen in this state. The translocational intermediate presented here represents a previously missing link in understanding the mechanism of translocation, revealing that the ribosome uses two distinct molecular ratchets, involving both intra- and intersubunit rotational movements, to drive the synchronous movement of tRNAs and mRNA.
authors: Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, 10117-Berlin, Germany.
authors: David J F
authors: DJ
authors: Ramrath
authors: Laura
authors: L
authors: Lancaster
authors: Thiemo
authors: T
authors: Sprink
authors: Thorsten
authors: T
authors: Mielke
authors: Justus
authors: J
authors: Loerke
authors: Harry F
authors: HF
authors: Noller
authors: Christian M T
authors: CM
authors: Spahn
mesh_terms: D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D007091:Image Processing, Computer-Assisted; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010441:Peptide Chain Elongation, Translational / Q000502:physiology*; D020653:Peptide Elongation Factor G / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D020653:Peptide Elongation Factor G; D012333:RNA, Messenger; D012343:RNA, Transfer
keywords: chimeric hybrid state; fusidic acid; ratcheting; ribosome dynamics; translocation mechanism