During protein synthesis, coupled translocation of messenger RNAs (mRNA) and transfer RNAs (tRNA) through the ribosome takes place following formation of each peptide bond. The reaction is facilitated by large-scale conformational changes within the ribosomal complex and catalyzed by elongtion factor G (EF-G). Previous structural analysis of the interaction of EF-G with the ribosome used either model complexes containing no tRNA or only a single tRNA, or complexes where EF-G was directly bound to ribosomes in the posttranslocational state. Here, we present a multiparticle cryo-EM reconstruction of a translocation intermediate containing two tRNAs trapped in transit, bound in chimeric intrasubunit ap/P and pe/E hybrid states. The downstream ap/P-tRNA is contacted by domain IV of EF-G and P-site elements within the 30S subunit body, whereas the upstream pe/E-tRNA maintains tight interactions with P-site elements of the swiveled 30S head. Remarkably, a tight compaction of the tRNA pair can be seen in this state. The translocational intermediate presented here represents a previously missing link in understanding the mechanism of translocation, revealing that the ribosome uses two distinct molecular ratchets, involving both intra-and intersubunit rotational movements, to drive the synchronous movement of tRNAs and mRNA.ratcheting | chimeric hybrid state | ribosome dynamics | translocation mechanism | fusidic acid D uring protein synthesis the ribosome iteratively incorporates new amino acids delivered by aminoacylated transfer RNAs (tRNA) into the growing polypeptide chain in a manner specified by the codons in a messenger RNAs (mRNA). This elongation cycle is controlled by the two translocational GTPases elongation factors (EF)-Tu and EF-G. Following EF-Tu-dependent delivery of aminoacyl-tRNA to the A site and peptide bond formation, the ribosome adopts a pretranslocational state containing a peptidyl A-site tRNA and a deacylated P-site tRNA. In the subsequent translocation reaction, the interplay between the ribosome and elongation factor EF-G shifts the tRNAs from the A and P sites to the P and E sites, respectively. In each of these binding sites a tRNA contacts both ribosomal subunits and interacts with the 30S and 50S subunits via its anticodon-stem loop (ASL) and acceptor arm, respectively (1). Partial tRNA movement can occur before the EF-G-dependent translocation step, involving spontaneous and reversible movement of the tRNA acceptor arms relative to the large ribosomal subunit, which leads to a shift from classic A/A and P/P binding states into intersubunit A/P and P/E hybrid states (where the first and second letters indicate tRNA contacts on the small and large subunits, respectively) (2-4).A remarkable feature of translocation is the precise coupling of movement of the tRNAs together with the bound mRNA (designated as the tRNA 2 •mRNA module), so that the mRNA advances by exactly one codon on the ribosome. Translocation is associated with large-scale conformational changes within the ribosomal complex,...
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 80S•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.
Researchers determine the structure of the ribosome-bound initiation factor 2.
Structures of macromolecules in their native state provide unique unambiguous insights into their functions. Cryo-electron tomography combined with subtomogram averaging demonstrated the power to solve such structures in situ at resolutions in the range of 3 Angstrom for some macromolecules. In order to be applicable to structural determination of the majority of macromolecules observable in cells in limited amounts, processing of tomographic data has to be performed in a high-throughput manner. Here we present TomoBEAR - a modular configurable workflow engine for streamlined processing of cryo-electron tomographic data for subtomogram averaging. TomoBEAR combines commonly used cryo-EM packages and reasonable presets to provide a transparent "white box" for data management and processing. We demonstrate applications of TomoBEAR to two datasets of purified proteins and to a membrane protein RyR1 in a membrane and demonstrate the ability to produce high resolution with minimal human intervention. TomoBEAR is an open-source and extendable package, it will accelerate the adoption of in situ structural biology by cryo-ET.
Eps15-homology domain containing proteins (EHDs) are eukaryotic, dynamin-related ATPases involved in cellular membrane trafficking. They oligomerize on membranes into filaments that induce membrane tubulation. While EHD crystal structures in open and closed conformations were previously reported, little structural information is available for the membrane-bound oligomeric form. Consequently, mechanistic insights into the membrane remodeling mechanism have remained sparse. Here, by using cryo-electron tomography and subtomogram averaging, we determined structures of nucleotide-bound EHD4 filaments on membrane tubes of various diameters at an average resolution of 7.6 Å. Assembly of EHD4 is mediated via interfaces in the G-domain and the helical domain. The oligomerized EHD4 structure resembles the closed conformation, where the tips of the helical domains protrude into the membrane. The variation in filament geometry and tube radius suggests a spontaneous filament curvature of approximately 1/70 nm−1. Combining the available structural and functional data, we suggest a model for EHD-mediated membrane remodeling.
25 (C.M.T.S.) 26 Kraushar ML, et al. Submission. 2 SUMMARY 27Protein synthesis must be finely tuned in the nervous system, as it represents an essential 28 feature of neurodevelopmental gene expression, and dominant pathology in neurological 29 disease. However, the architecture of ribosomal complexes in the developing mammalian brain 30 has not been analyzed at high resolution. This study investigates the architecture of ribosomes 31 ex vivo from the embryonic and perinatal mouse neocortex, revealing Ebp1 as a 60S peptide 32 tunnel exit binding factor at near-atomic resolution by multiparticle cryo-electron microscopy. 33 The impact of Ebp1 on the neuronal proteome was analyzed by pSILAC and BONCAT coupled 34 mass spectrometry, implicating Ebp1 in neurite outgrowth proteostasis, with in vivo embryonic 35 Ebp1 knockdown resulting in dysregulation of neurite outgrowth. Our findings reveal Ebp1 as a 36 central component of neocortical protein synthesis, and the 60S peptide tunnel exit as a focal 37 point of gene expression control in the molecular specification of neuronal morphology. 38 39
Dynamin-related Eps15-homology domain containing proteins (EHDs) oligomerize on membrane surfaces into filaments leading to membrane remodeling. EHD crystal structures in an open and a closed conformation were previously reported, but structural information on the membrane-bound EHD oligomeric structure has remained enigmatic. Consequently, mechanistic insight into EHD-mediated membrane remodeling is lacking. Here, by using cryo-electron tomography and subtomogram averaging, we determined the structure of an EHD4 filament on a tubular membrane template at an average resolution of 7.6 Å. Assembly of EHD4 is mediated via interfaces in the G-domain and the helical domain. The oligomerized EHD4 structure resembles the closed conformation, where the tips of the helical domains protrude into the membrane. The variation in filament geometry and tube radius suggests the AMPPNP-bound filament has a spontaneous curvature of approximately 1/70 nm-1. Combining the available structural and functional data, we propose a model of EHD-mediated membrane remodeling.
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