RESUMO
Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex vivo-derived E. coli polysomes in the PURE in vitro translation system and analyzed the actively elongating polysomes by cryo-EM. We find that 31% of 70S ribosomes assemble into disome complexes that represent eight distinct functional states including decoding and termination intermediates, and a pre-nucleophilic attack state. The functional diversity of disome complexes together with RNase digest experiments suggests that paused disome complexes transiently form during ongoing elongation. Structural analysis revealed five disome interfaces between leading and queueing ribosomes that undergo rearrangements as the leading ribosome traverses through the elongation cycle. Our findings reveal at the molecular level how bL9's CTD obstructs the factor binding site of queueing ribosomes to thwart harmful collisions and illustrate how translation dynamics reshape inter-ribosomal contacts.
Assuntos
Escherichia coli , Ribossomos , Escherichia coli/genética , Escherichia coli/química , Microscopia Crioeletrônica , Ribossomos/metabolismo , Biossíntese de Proteínas , Polirribossomos/metabolismoRESUMO
Translocation moves the tRNA2â mRNA module directionally through the ribosome during the elongation phase of protein synthesis. Although translocation is known to entail large conformational changes within both the ribosome and tRNA substrates, the orchestrated events that ensure the speed and fidelity of this critical aspect of the protein synthesis mechanism have not been fully elucidated. Here, we present three high-resolution structures of intermediates of translocation on the mammalian ribosome where, in contrast to bacteria, ribosomal complexes containing the translocase eEF2 and the complete tRNA2â mRNA module are trapped by the non-hydrolyzable GTP analog GMPPNP. Consistent with the observed structures, single-molecule imaging revealed that GTP hydrolysis principally facilitates rate-limiting, final steps of translocation, which are required for factor dissociation and which are differentially regulated in bacterial and mammalian systems by the rates of deacyl-tRNA dissociation from the E site.
Assuntos
Guanosina Trifosfato/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Animais , Bactérias/metabolismo , Guanosina Trifosfato/química , Humanos , Hidrólise , Sítios Internos de Entrada Ribossomal , Mamíferos/metabolismo , Modelos Moleculares , Fator 2 de Elongação de Peptídeos/química , Fator 2 de Elongação de Peptídeos/metabolismo , Domínios Proteicos , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA de Transferência/química , Ribossomos/químicaRESUMO
Internal ribosomal entry sites (IRESs) are structured cis-acting RNAs that drive an alternative, cap-independent translation initiation pathway. They are used by many viruses to hijack the translational machinery of the host cell. IRESs facilitate translation initiation by recruiting and actively manipulating the eukaryotic ribosome using only a subset of canonical initiation factor and IRES transacting factors. Here we present cryo-EM reconstructions of the ribosome 80S- and 40S-bound Hepatitis C Virus (HCV) IRES. The presence of four subpopulations for the 80Sâ¢HCV IRES complex reveals dynamic conformational modes of the complex. At a global resolution of 3.9 Å for the most stable complex, a derived atomic model reveals a complex fold of the IRES RNA and molecular details of its interaction with the ribosome. The comparison of obtained structures explains how a modular architecture facilitates mRNA loading and tRNA binding to the P-site. This information provides the structural foundation for understanding the mechanism of HCV IRES RNA-driven translation initiation.
Assuntos
Sítios Internos de Entrada Ribossomal , RNA Viral/química , Subunidades Ribossômicas/química , Sequência de Aminoácidos , Sequência de Bases , Hepatite C/metabolismo , Humanos , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Ligação ProteicaRESUMO
Elongation factor G (EF-G) catalyzes tRNA translocation on the ribosome. Here a cryo-EM reconstruction of the 70S*EF-G ribosomal complex at 7.3 A resolution and the crystal structure of EF-G-2*GTP, an EF-G homolog, at 2.2 A resolution are presented. EF-G-2*GTP is structurally distinct from previous EF-G structures, and in the context of the cryo-EM structure, the conformational changes are associated with ribosome binding and activation of the GTP binding pocket. The P loop and switch II approach A2660-A2662 in helix 95 of the 23S rRNA, indicating an important role for these conserved bases. Furthermore, the ordering of the functionally important switch I and II regions, which interact with the bound GTP, is dependent on interactions with the ribosome in the ratcheted conformation. Therefore, a network of interaction with the ribosome establishes the active GTP conformation of EF-G and thus facilitates GTP hydrolysis and tRNA translocation.
Assuntos
Guanosina Trifosfato/metabolismo , Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Thermus thermophilus/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Guanilil Imidodifosfato/metabolismo , Modelos Moleculares , Fator G para Elongação de Peptídeos/ultraestrutura , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ribossomos/ultraestrutura , Relação Estrutura-AtividadeRESUMO
Internal ribosome entry sites (IRESs) facilitate an alternative, end-independent pathway of translation initiation. A particular family of dicistroviral IRESs can assemble elongation-competent 80S ribosomal complexes in the absence of canonical initiation factors and initiator transfer RNA. We present here a cryo-EM reconstruction of a dicistroviral IRES bound to the 80S ribosome. The resolution of the cryo-EM reconstruction, in the subnanometer range, allowed the molecular structure of the complete IRES in its active, ribosome-bound state to be solved. The structure, harboring three pseudoknot-containing domains, each with a specific functional role, shows how defined elements of the IRES emerge from a compactly folded core and interact with the key ribosomal components that form the A, P and E sites, where tRNAs normally bind. Our results exemplify the molecular strategy for recruitment of an IRES and reveal the dynamic features necessary for internal initiation.