Protein biosynthesis, the translation of the genetic code into polypeptides, occurs on ribonucleoprotein particles called ribosomes. Although X-ray structures of bacterial ribosomes are available, high-resolution structures of eukaryotic 80S ribosomes are lacking. Using cryoelectron microscopy and single-particle reconstruction, we have determined the structure of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution. This map, together with a 6.1-Å map of a Saccharomyces cerevisiae 80S ribosome, has enabled us to model ∼98% of the rRNA. Accurate assignment of the rRNA expansion segments (ES) and variable regions has revealed unique ES-ES and r-protein-ES interactions, providing insight into the structure and evolution of the eukaryotic ribosome.modeling | molecular dynamics | flexible fitting I n all living cells, the translation of mRNA into polypeptide occurs on ribosomes. Ribosomes provide a platform upon which aminoacyl-tRNAs interact with the mRNA as well as position the aminoacyl-tRNAs for peptide-bond formation (1). Ribosomes are composed of two subunits, a small subunit that monitors the mRNA-tRNA codon-anticodon duplex to ensure fidelity of decoding (2, 3) and a large subunit that contains the active site where peptide-bond formation occurs (4). Both the small and large subunits are composed of RNA and protein: In eubacteria such as Escherichia coli, the small subunit contains one 16S rRNA and 21 ribosomal proteins (r proteins), whereas the large subunit contains 5S and 23S rRNAs and 33 r proteins. Crystal structures of the complete bacterial 70S ribosome were initially reported at 5.5 Å (5), with an interpretation based on atomic models of the individual subunit structures (6-8), and are now available at atomic resolution (9). These structures have provided unparalleled insight into the mechanism of different steps of translation (1) as well as inhibition by antibiotics (10).Compared to the bacterial ribosome, the eukaryotic counterpart is more complicated, containing expansion segments (ES) and variable regions in the rRNA as well as many additional r proteins and r-protein extensions. Plant and fungal 80S ribosomes contain ∼5;500 nucleotides (nts) of rRNA and ∼80 r proteins, whereas bacterial 70S ribosomes comprise ∼4;500 nts and 54 r proteins. The additional elements present in eukaryotic ribosomes may reflect the increased complexity of translation regulation in eukaryotic cells, as evident for assembly, translation initiation, and development, as well as the phenomenon of localized translation (11-15).Early models for eukaryotic ribosomes were derived from electron micrographs of negative-stain or freeze-dried ribosomal particles (16) and localization of r proteins was attempted using immuno-EM and cross-linking approaches; see, for example, refs. 17-20. The first cryo-EM reconstruction of a eukaryotic 80S ribosome was reported for wheat germ (Triticum aestivum) at 38 Å (21). Initial core models for the yeast 80S ribosome were built at 15-Å resolution (22) by docking the rRNA s...

Protein synthesis in all living organisms occurs on ribonucleoprotein particles, called ribosomes. Despite the universality of this process, eukaryotic ribosomes are significantly larger in size than their bacterial counterparts due in part to the presence of 80 r proteins rather than 54 in bacteria. Using cryoelectron microscopy reconstructions of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution, together with a 6.1-Å map of a translating Saccharomyces cerevisiae 80S ribosome, we have localized and modeled 74∕80 (92.5%) of the ribosomal proteins, encompassing 12 archaeal/eukaryote-specific small subunit proteins as well as the complete complement of the ribosomal proteins of the eukaryotic large subunit. Near-complete atomic models of the 80S ribosome provide insights into the structure, function, and evolution of the eukaryotic translational apparatus.homology modeling | RNA | translation | flexible fitting | molecular dynamics P rotein synthesis occurs on large macromolecular complexes, called ribosomes (1). Ribosomes are composed of two subunits, both of which are built from protein and RNA. Bacterial ribosomes, for example, in Escherichia coli, contain a small subunit composed of one 16S rRNA and 21 ribosomal proteins (r proteins), and a large subunit containing 5S and 23S rRNAs and 33 r proteins. In contrast, eukaryotic ribosomes are much larger and more complex, containing additional RNA in the form of so-called expansion segments (ES) as well as many additional r proteins and r-protein extensions. The additional r proteins present in eukaryotic ribosomes are likely to reflect the increased complexity of translation regulation in eukaryotic cells (2-5). Moreover, many of these eukaryote-specific components have been associated with human disorders (4). Thus, structural insight into the localization of these elements will be important to furthering our understanding of eukaryotic translation regulation as well as disease.Compared with the ∼54 r proteins of the bacterial ribosome, plant and fungal 80S ribosomes contain ∼80 r proteins (see Table S1 for r-protein nomenclature). Crystal structures have revealed the location of each small and large subunit r protein within bacterial ribosomes (6-12) as well as the r proteins within the archaeal large ribosomal subunit (13,14). In contrast, the localization of ribosomal proteins within eukaryotic 80S ribosomes has come mainly from early studies using immuno-EM and cross-linking approaches (see, for example, refs. 15-18). Moreover, the first molecular models for the eukaryotic ribosome were built at 15-Å resolution by docking the structures of the bacterial small 30S subunit (6) and archaeal large 50S subunit (13), thus only identifying the location of a total of 46 eukaryotic r proteins with bacterial or archaeal homologues (19). Recently, cryo-EM reconstructions of plant and fungal 80S ribosomes have led to the localization of three eukaryote-specific r proteins: RACK1 (20) and S19e (21) in the small subunit and L30e in the large subunit...

In all living cells, protein synthesis occurs on ribonucleoprotein particles called ribosomes. Molecular models have been reported for complete bacterial 70S and eukaryotic 80S ribosomes; however, only molecular models of large 50S subunits have been reported for archaea. Here, we present a complete molecular model for the Pyrococcus furiosus 70S ribosome based on a 6.6 Å cryo-electron microscopy map. Moreover, we have determined cryo-electron microscopy reconstructions of the Euryarchaeota Methanococcus igneus and Thermococcus kodakaraensis 70S ribosomes and Crenarchaeota Staphylothermus marinus 50S subunit. Examination of these structures reveals a surprising promiscuous behavior of archaeal ribosomal proteins: We observe intersubunit promiscuity of S24e and L8e (L7ae), the latter binding to the head of the small subunit, analogous to S12e in eukaryotes. Moreover, L8e and L14e exhibit intrasubunit promiscuity, being present in two copies per archaeal 50S subunit, with the additional binding site of L14e analogous to the related eukaryotic r-protein L27e. Collectively, these findings suggest insights into the evolution of eukaryotic ribosomal proteins through increased copy number and binding site promiscuity.