RESUMO
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 80Sâ CrPV-STOP â eRF1 â eRF3 â GMPPNP and 80Sâ CrPV-STOP â 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 â eRF3 â 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.
Assuntos
Dicistroviridae/genética , Fatores de Terminação de Peptídeos/metabolismo , RNA Mensageiro/química , RNA Viral/química , Ribossomos/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Microscopia Crioeletrônica/métodos , Dicistroviridae/química , Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/metabolismo , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fatores de Terminação de Peptídeos/química , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , RNA Viral/metabolismo , Ribossomos/químicaRESUMO
Some viruses exploit internal initiation for their propagation in the host cell. This type of initiation is facilitated by structured elements (internal ribosome entry site, IRES) upstream of the initiator AUG and requires only a reduced number of canonical initiation factors. An important example are IRES of the virus family Dicistroviridae that bind to the inter-subunit side of the small ribosomal 40S subunit and lead to the formation of elongation-competent 80S ribosomes without the help of any initiation factor. Here, we present a comprehensive functional and structural analysis of eukaryotic-specific ribosomal protein rpS25 in the context of this type of initiation and propose a structural model explaining the essential involvement of rpS25 for hijacking the ribosome.