Cryo-EM of Ribosomal 80S Complexes with Termination Factors Reveals the Translocated Cricket Paralysis Virus IRES

• Published in: Molecular cell 2015-02, Vol.57 (3), p.422-432
• Main Authors: Muhs, Margarita, Hilal, Tarek, Mielke, Thorsten, Skabkin, Maxim A., Sanbonmatsu, Karissa Y., Pestova, Tatyana V., Spahn, Christian M.T.
• Format: Article
• Language: English
• Subjects: ATP-Binding Cassette Transporters - metabolism; Cryoelectron Microscopy - methods; Dicistroviridae - chemistry; Dicistroviridae - genetics; Eukaryotic Initiation Factors - chemistry; Eukaryotic Initiation Factors - metabolism; Models, Molecular; Molecular Conformation; Molecular Sequence Data; Nucleic Acid Conformation; Peptide Termination Factors - chemistry; Peptide Termination Factors - metabolism; Protein Biosynthesis; Ribosomes - chemistry; Ribosomes - metabolism; RNA, Messenger - chemistry; RNA, Messenger - metabolism; RNA, Transfer - chemistry; RNA, Transfer - metabolism; RNA, Viral - chemistry; RNA, Viral - metabolism
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2014.12.016

The cricket paralysis virus (CrPV) uses an internal ribosomal entry site (IRES) to hijack the ribosome. In a remarkable RNA-based mechanism involving neither initiation factor nor initiator tRNA, the CrPV IRES jumpstarts translation in the elongation phase from the ribosomal A site. Here, we present cryoelectron microscopy (cryo-EM) maps of 80S⋅CrPV-STOP⋅eRF1⋅eRF3⋅GMPPNP and 80S⋅CrPV-STOP⋅eRF1 complexes, revealing a previously unseen binding state of the IRES and directly rationalizing that an eEF2-dependent translocation of the IRES is required to allow the first A-site occupation. During this unusual translocation event, the IRES undergoes a pronounced conformational change to a more stretched conformation. At the same time, our structural analysis provides information about the binding modes of eRF1⋅eRF3⋅GMPPNP and eRF1 in a minimal system. It shows that neither eRF3 nor ABCE1 are required for the active conformation of eRF1 at the intersection between eukaryotic termination and recycling. [Display omitted] •The CrPV IRES is directly visualized in the post-translocated state•During pseudo-translocation, the IRES undergoes a pronounced conformational change•Factors eRF1 and eRF1/eRF3 are visualized on the ribosome in a minimal system•eRF1 alone can adopt the active conformation on the ribosome The unusual mechanism by which CrPV IGR IRES initiates protein synthesis includes a pseudo-translocation step. Here, cryoelectron microscopy reveals the state of the CrPV IGR IRES on the ribosome after translocation, providing insight into the RNA-based mechanism of internal translation initiation.