The triplet-based genetic code requires that translating ribosomes maintain the reading frame of a messenger RNA faithfully to ensure correct protein synthesis¹. However, in programmed -1 ribosomal frameshifting², a specific subversion of frame maintenance takes place, wherein the ribosome is forced to shift one nucleotide backwards into an overlapping reading frame and to translate an entirely new sequence of amino acids. This process is indispensable in the replication of numerous viral pathogens, including HIV and the coronavirus associated with severe acute respiratory syndrome³, and is also exploited in the expression of several cellular genes⁴. Frameshifting is promoted by an mRNA signal composed of two essential elements: a heptanucleotide 'slippery' sequence⁵ and an adjacent mRNA secondary structure, most often an mRNA pseudoknot⁶. How these components operate together to manipulate the ribosome is unknown. Here we describe the observation of a ribosome–mRNA pseudoknot complex that is stalled in the process of -1 frameshifting. Cryoelectron microscopic imaging of purified mammalian 80S ribosomes from rabbit reticulocytes paused at a coronavirus pseudoknot reveals an intermediate of the frameshifting process. From this it can be seen how the pseudoknot interacts with the ribosome to block the mRNA entrance channel, compromising the translocation process and leading to a spring-like deformation of the P-site transfer RNA. In addition, we identify movements of the likely eukaryotic ribosomal helicase and confirm a direct interaction between the translocase eEF2 and the P-site tRNA. Together, the structural changes provide a mechanical explanation of how the pseudoknot manipulates the ribosome into a different reading frame.

1. Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
2. Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK
3. Oxford Centre for Molecular Sciences, Central Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QH, UK
4. †Present address: Institut de Génétique et Microbiologie, UMR8621, Université Paris-Sud, 91405 Orsay, France
5. *These authors contributed equally to this work

Correspondence to: Robert J. C. Gilbert^2,3Ian Brierley¹ Correspondence and requests for materials should be addressed to I.B. (Email: ib103@mole.bio.cam.ac.uk) or R.J.C.G. (Email: gilbert@strubi.ox.ac.uk). Electron-density maps have been deposited in the European Bioinformatics Institute Electron Microscopy database, accession numbers EMD-1197, EMD-1198 and EMD-1199 (www.ebi.ac.uk/msd/iims/3D_EMdep.html).

Received 3 February 2006 |Accepted 20 March 2006

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