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.

Plastid-specific ribosomal proteins (PSRPs) have been proposed to play roles in the light-dependent regulation of chloroplast translation. Here we demonstrate that PSRP1 is not a bona fide ribosomal protein, but rather a functional homologue of the Escherichia coli cold-shock protein pY. Three-dimensional Cryo-electron microscopic (Cryo-EM) reconstructions reveal that, like pY, PSRP1 binds within the intersubunit space of the 70S ribosome, at a site overlapping the positions of mRNA and A-and P-site tRNAs. PSRP1 induces conformational changes within ribosomal components that comprise several intersubunit bridges, including bridge B2a, thereby stabilizes the ribosome against dissociation. We find that the presence of PSRP1/pY lowers the binding of tRNA to the ribosome. Furthermore, similarly to tRNAs, PSRP1/pY is recycled from the ribosome by the concerted action of the ribosome-recycling factor (RRF) and elongation factor G (EF-G). These results suggest a novel function for EF-G and RRF in the post-stress return of PSRP1/pY-inactivated ribosomes to the actively translating pool.Chloroplasts are intracellular organelles present in higher plants and algae; they contain the entire machinery with which the process of photosynthesis is conducted. According to the endosymbiotic theory of chloroplast evolution (1-3), this organelle originated through engulfment of a photosynthetic unicellular prokaryote by a eukaryotic host cell, and the subsequent integration of the two genomes (that of the engulfed prokaryote, and the eukaryotic nucleus) through a process of gene transfers from the chloroplast to the nuclear genome. Thus, although the chloroplast carries its own transcriptional and translational machineries, the development and maintenance of the chloroplast are dependent on the coordinated expression of chloroplast-and nuclear-encoded gene products.The light-dependent process of photosynthesis is the primary function of the chloroplast. Because the components that are crucial for the biogenesis of the photosynthetic apparatus are encoded by both the chloroplast and nuclear genomes, the plant cell has evolved several mechanisms to achieve concerted regulation of gene expression in the two cellular compartments, in response to changes in illumination (4 -6). Regulation of gene expression is primarily post-transcriptional, and is achieved through altered mRNA processing and stability, and the control of the translational apparatus itself in response to environmental signals like light (7-9). It has been demonstrated that the redox state of the chloroplast achieved in response to photosynthetic electron transport can regulate protein synthesis within the chloroplast at the stages of initiation and elongation (10 -12).A detailed analysis of the chloroplast translational machinery, the chloroplast ribosome together with its trans-acting translational factors, will provide important clues as to how such gene regulation is achieved. Proteomic characterization of the chloroplast ribosomes (chlororibosomes) from spinach has r...

Translation of specific small peptides on the ribosome can confer resistance to macrolide antibiotics. To reveal the molecular details of this and related phenomena, stable RNA-peptide conjugates that mimic peptidyl-tRNA would be desirable, especially for ribosome structural biology. A flexible solid-phase synthesis strategy now allows efficient access to these highly requested derivatives without restriction on the RNA and peptide sequences.

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Maintenance of the translation reading frame is one of the most remarkable achievements of the ribosome while decoding the information of an mRNA. Loss of the reading frame through spontaneous frameshifting occurs with a frequency of one in 30,000 amino acid incorporations. However, at many recoding sites, the mechanism that controls reading frame maintenance is switched off. One such example is the programmed +1 frameshift site of the prfB gene encoding the termination factor RF2, in which slippage into the forward frame by one nucleotide can attain an efficiency of approximately 100%, namely, four orders of magnitude higher than normally observed. Here, using the RF2 frameshift window, we demonstrate that premature release of the E site tRNA from the ribosome is coupled with high-level frameshifting. Consistently, in a minimal system, the presence of the E site tRNA prevents the +1 frameshift event, illustrating the importance of the E site for reading-frame maintenance.

During translation, usually only one in Ϸ400 misincorporations affects the function of a nascent protein, because only chemically similar near-cognate amino acids are misincorporated in place of the cognate one. The deleterious misincorporation of a chemically dissimilar noncognate amino acid during the selection process is precluded by the presence of a tRNA at the ribosomal E-site. However, the selection of first aminoacyl-tRNA, directly after initiation, occurs without an occupied E-site, i.e., when only the P-site is filled with the initiator tRNA and thus should be highly error-prone. Here, we show how bacterial ribosomes have solved this accuracy problem: In the absence of a Shine-Dalgarno (SD) sequence, the first decoding step at the A-site after initiation is extremely error-prone, even resulting in the significant incorporation of noncognate amino acids. In contrast, when a SD sequence is present, the incorporation of noncognate amino acids is not observed. This is precisely the effect that the presence of a cognate tRNA at the E-site has during the elongation phase. These findings suggest that during the initiation phase, the SD interaction functionally compensates for the lack of codon-anticodon interaction at the E-site by reducing the misincorporation of near-cognate amino acids and prevents noncognate misincorporation. E-site ͉ translational errorsT he binding of aminoacyl-tRNAs to the ribosome is dictated by the complementarity between the anticodon of the tRNA and the codon of the mRNA. To ensure the high fidelity of translation, the correct stereochemistry of the mRNA-tRNA codon-anticodon interaction is monitored by components of the small ribosomal subunit in a process known as decoding (reviewed in ref. 1). During decoding, the first and second nucleotide positions (in terms of the codon) of the mRNA-tRNA duplex are closely monitored, whereas interaction at the third or wobble position is less strictly recognized. Consistently, the misincorporation of the wrong amino acids into polypeptide chains usually occurs through the binding of nearcognate aminoacyl-tRNAs, i.e., those tRNAs carrying an anticodon similar to that of the cognate aminoacyl-tRNA, rather than noncognate aminoacyl-tRNAs, which carry dissimilar anticodons. Nascent polypeptide chains are surprisingly tolerant to misincorporation, with only one in Ϸ400 misincorporations being deleterious for the protein's activity (reviewed in ref.2). The reason for this is that usually near-cognate aminoacyl-tRNAs are selected instead of the cognate aminoacyl-tRNA, and the genetic code lexicon is organized in such a way that near-cognate tRNAs bear amino acids that are chemically similar to those carried by the cognate tRNA. For example, the misincorporation of an aspartate (codon: GAU/C) by near-cognate Asp-tRNA, instead of glutamate (GAA/G) by the cognate Glu-tRNA, both incorporate acidic amino acids. The middle and, in most cases, the first position of a codon are almost never misread, even under error-inducing conditions such as high magnesiu...