Direct observation of distinct A/P hybrid-state tRNAs in translocating ribosomes.
Transfer RNAs (tRNAs) link the genetic code in the form of messenger RNA (mRNA) to protein sequence. Translocation of tRNAs through the ribosome from aminoacyl (A) site to peptidyl (P) site and from P site to exit site is catalyzed in eukaryotes by the translocase elongation factor 2 (EF-2) and in prokaryotes by its homolog EF-G. During tRNA movement one or more "hybrid" states (A/P) is occupied, but molecular details of them and of the translocation process are limited. Here we show by cryo-electron microscopy that a population of mammalian ribosomes stalled at an mRNA pseudoknot structure contains structurally distorted tRNAs in two different A/P hybrid states. In one (A/P'), the tRNA is in contact with the translocase EF-2, which induces it. In the other (A/P''), the translocase is absent. The existence of these alternative A/P intermediate states has relevance to our understanding of the mechanics and kinetics of translocation.
Flanagan
John F
JF
Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom.
Namy
Olivier
O
Institut de Genetique et Microbiologie, Universite Paris-Sud, batiment 400, 91405 Orsay Cedex, France.
Brierley
Ian
I
Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom.
Gilbert
Robert J C
RJC
Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom.
D001483:Base Sequence; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020836:Protein Structure, Quaternary; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism
D046911:Macromolecular Substances; D012343:RNA, Transfer