title: A new tRNA intermediate revealed on the ribosome during EF4-mediated back-translocation.
abstract: EF4 (LepA) is an almost universally conserved translational GTPase in eubacteria. It seems to be essential under environmental stress conditions and has previously been shown to back-translocate the tRNAs on the ribosome, thereby reverting the canonical translocation reaction. In the current work, EF4 was directly visualized in the process of back-translocating tRNAs by single-particle cryo-EM. Using flexible fitting methods, we built a model of ribosome-bound EF4 based on the cryo-EM map and a recently published unbound EF4 X-ray structure. The cryo-EM map establishes EF4 as a noncanonical elongation factor that interacts not only with the elongating ribosome, but also with the back-translocated tRNA in the A-site region, which is present in a previously unseen, intermediate state and deviates markedly from the position of a canonical A-tRNA. Our results, therefore, provide insight into the underlying structural principles governing back-translocation.
authors.affiliation: Institut für Medizinische Physik und Biophysik, Charite-Universitätsmedizin Berlin, Ziegelstrasse 5-9, 10117-Berlin, Germany.
authors.forename: Sean R
authors.initials: SR
authors.lastname: Connell
authors.forename: Maya
authors.initials: M
authors.lastname: Topf
authors.forename: Yan
authors.initials: Y
authors.lastname: Qin
authors.forename: Daniel N
authors.initials: DN
authors.lastname: Wilson
authors.forename: Thorsten
authors.initials: T
authors.lastname: Mielke
authors.forename: Paola
authors.initials: P
authors.lastname: Fucini
authors.forename: Knud H
authors.initials: KH
authors.lastname: Nierhaus
authors.forename: Christian M T
authors.initials: CM
authors.lastname: Spahn
mesh_terms: D001693:Biological Transport, Active; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D046911:Macromolecular Substances; D008958:Models, Molecular; D010448:Peptide Initiation Factors; D012329:RNA, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism*; D035602:Transcriptional Elongation Factors / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C515651:LepA protein, E coli; D046911:Macromolecular Substances; D010448:Peptide Initiation Factors; D012329:RNA, Bacterial; D035602:Transcriptional Elongation Factors; D006160:Guanosine Triphosphate; D012343:RNA, Transfer