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Crystal structures of prokaryotic ribosomes have described in detail the universally conserved core of the translation mechanism. However, many facets of the translation process in eukaryotes are not shared with prokaryotes. The crystal structure of the yeast 80S ribosome determined at 4.15 angstrom resolution reveals the higher complexity of eukaryotic ribosomes, which are 40% larger than their bacterial counterparts. Our model shows how eukaryote-specific elements considerably expand the network of interactions within the ribosome and provides insights into eukaryote-specific features of protein synthesis. Our crystals capture the ribosome in the ratcheted state, which is essential for translocation of mRNA and transfer RNA (tRNA), and in which the small ribosomal subunit has rotated with respect to the large subunit. We describe the conformational changes in both ribosomal subunits that are involved in ratcheting and their implications in coordination between the two associated subunits and in mRNA and tRNA translocation. 10.1126/science.1194294

We report the crystal structure of release factor 2 bound to ribosome with an aminoacyl tRNA substrate analog at the ribosomal P site, at 3.1 [Angstrom] resolution. The structure shows that upon stop-codon recognition, the universally conserved GGQ motif packs tightly into the peptidyl transferase center. Nucleotide A2602 of 23S rRNA, implicated in peptide release, packs with the GGQ motif in release factor 2. The ribose of A76 of the peptidyl-tRNA adopts the C2'-endo conformation, and the 2' hydroxyl of A76 is within hydrogen-bond distance of the 2' hydroxyl of A2451. The structure suggests how a catalytic water can be coordinated in the peptidyl transferase center and, together with previous biochemical and computational data, suggests a model for how the ester bond between the peptidyl tRNA and the nascent peptide is hydrolyzed. ribosome structure | translational termination | X-ray crystallography doi/10.1073/pnas.1003995107

The means by which a polypeptide chain acquires its unique 3-D structure is a fundamental question in biology. During its synthesis on the ribosome, a nascent chain (NC) emerges vectorially and will begin to fold in a cotranslational fashion. The complex environment of the cell, coupled with the gradual emergence of the ribosome-tethered NC during its synthesis, imposes conformational restraints on its folding landscape that differ from those placed on an isolated protein when stimulated to fold following denaturation in solution. To begin to examine cotranslational folding as it would occur within a cell, we produce highly selective, isotopically labeled NCs bound to isotopically silent ribosomes in vivo. We then apply NMR spectroscopy to study, at a residue specific level, the conformation of NCs consisting of different fractional lengths of the polypeptide chain corresponding to a given protein. This combined approach provides a powerful means of generating a series of snapshots of the folding of the NC as it emerges from the ribosome. Application of this strategy to the NMR analysis of the progressive synthesis of an Ig-like domain reveals the existence of a partially folded ribosome-bound species that is likely to represent an intermediate species populated during the cotranslational folding process. cotranslational folding | ribosome | SecM doi/10.1073/pnas.0903750106

Elongation factor G (EF-G) is a guanosine triphosphatase (GTPase) that plays a crucial role in the translocation of transfer RNAs (tRNAs) and messenger RNA (mRNA) during translation by the ribosome. We report a crystal structure refined to 3.6 angstrom resolution of the ribosome trapped with EF-G in the posttranslocationat state using the antibiotic fusidic acid. Fusidic acid traps EF-G in a conformation intermediate between the guanosine triphosphate and guanosine diphosphate forms. The interaction of EF-G with ribosomal elements implicated in stimulating catalysis, such as the L10-L12 stalk and the L11 region, and of domain IV of EF-G with the tRNA at the peptidyl-tRNA binding site (P site) and with mRNA shed light on the role of these elements in EF-G function. The stabilization of the mobile stalks of the ribosome also results in a more complete description of its structure. 10.1126/science.1179709

The ribosome selects a correct transfer RNA (tRNA) for each amino acid added to the polypeptide chain, as directed by messenger RNA. Aminoacyl-tRNA is delivered to the ribosome by elongation factor Tu (EF-Tu), which hydrolyzes guanosine triphosphate (GTP) and releases tRNA in response to codon recognition. The signaling pathway that leads to GTP hydrolysis upon codon recognition is critical to accurate decoding. Here we present the crystal structure of the ribosome complexed with EF-Tu and aminoacyl-tRNA, refined to 3.6 angstrom resolution. The structure reveals details of the tRNA distortion that allows aminoacyl-tRNA to interact simultaneously with the decoding center of the 30S subunit and EF-Tu at the factor binding site. A series of conformational changes in EF-Tu and aminoacyl-tRNA suggests a communication pathway between the decoding center and the guanosine triphosphatase center of EF-Tu. 10.1126/science.1179700

Protein biosynthesis on the ribosome requires repeated cycles of ratcheting, which couples rotation of the two ribosomal subunits with respect to each other, and swiveling of the head domain of the small subunit. However, the molecular basis for how the two ribosomal subunits rearrange contacts with each other during ratcheting while remaining stably associated is not known. Here, we describe x-ray crystal structures of the intact Escherichia coli ribosome, either in the apo-form (3.5 angstrom resolution) or with one (4.0 angstrom resolution) or two (4.0 angstrom resolution) anticodon stem-loop tRNA mimics bound, that reveal intermediate states of intersubunit rotation. In the structures, the interface between the small and large ribosomal subunits rearranges in discrete steps along the ratcheting pathway. Positioning of the head domain of the small subunit is controlled by interactions with the large subunit and with the tRNA bound in the peptidyl-tRNA site. The intermediates observed here provide insight into how tRNAs move into the hybrid state of binding that precedes the final steps of mRNA and tRNA translocation.

The Leishmania tarentolae mitochondrial ribosome (Lmr) is a minimal ribosomal RNA (rRNA)-containing ribosome. We have obtained a cryo-EM map of the Lmr. The map reveals several features that have not been seen in previously-determined structures of eubacterial or eukaryotic (cytoplasmic or organellar) ribosomes to our knowledge. Comparisons of the Lmr map with X-ray crystallographic and cryo-EM maps of the eubacterial ribosomes and a cryo-EM map of the mammalian mitochondrial ribosome show that (i) the overall structure of the Lmr is considerably more porous, (ii) the topology of the intersubunit space is significantly different, with fewer intersubunit bridges, but more tunnels, and (iii) several of the functionally-important rRNA regions, including the [alpha]-sarcin-ricin loop, have different relative positions within the structure. Furthermore, the major portions of the mRNA channel, the tRNA passage, and the nascent polypeptide exit tunnel contain Lmr-specific proteins, suggesting that the mechanisms for mRNA recruitment, tRNA interaction, and exiting of the nascent polypeptide in Lmr must differ markedly from the mechanisms deduced for ribosomes in other organisms. Our study identifies certain structural features that are characteristic solely of mitochondrial ribosomes and other features that are characteristic of both mitochondrial and chloroplast ribosomes (i.e., organellar ribosomes). cryo-EM | Leishmania tarentolae | organellar ribosome

We report the crystal structure of a translation termination complex formed by the Thermus thermophilus 70S ribosome bound with release factor RF2, in response to a UAA stop codon, solved at 3 [Angstrom] resolution. The backbone of helix [alpha]5 and the side chain of serine of the conserved SPF motif of RF2 recognize U1 and A2 of the stop codon, respectively. A3 is unstacked from the first 2 bases, contacting Thr-216 and Val-203 of RF2 and stacking on G530 of 16S rRNA. The structure of the RF2 complex supports our previous proposal that conformational changes in the ribosome in response to recognition of the stop codon stabilize rearrangement of the switch loop of the release factor, resulting in docking of the universally conserved GGQ motif in the PTC of the 50S subunit. As seen for the RF1 complex, the main-chain amide nitrogen of glutamine in the GGQ motif is positioned to contribute directly to catalysis of peptidyl-tRNA hydrolysis, consistent with mutational studies, which show that most side-chain substitutions of the conserved glutamine have little effect. We show that when the H-bonding capability of the main-chain N-H of the conserved glutamine is eliminated by substitution with proline, peptidyl-tRNA esterase activity is abolished, consistent with its proposed role in catalysis. 70S ribosome structure | stop codon recognition | polypeptide release

During protein synthesis, tRNAs and mRNA move through the ribosome between aminoacyl (A), peptidyl (P), and exit (E) sites of the ribosome in a process called translocation. Translocation is accompanied by the displacement of the tRNAs on the large ribosomal subunit toward the hybrid A/P and P/E states and by a rotational movement (ratchet) of the ribosomal subunits relative to one another. So far, the structure of the ratcheted state has been observed only when translation factors were bound to the ribosome. Using cryo-electron microscopy and classification, we show here that ribosomes can spontaneously adopt a ratcheted conformation with tRNAs in their hybrid states. The peptidyl-tRNA molecule in the A/P state, which is visualized here, is not distorted compared with the A/A state except for slight adjustments of its acceptor end, suggesting that the displacement of the A-site tRNA on the 50S subunit is passive and is induced by the 30S subunit rotation. Simultaneous subunit ratchet and formation of the tRNA hybrid states precede and may promote the subsequent rapid and coordinated tRNA translocation on the 30S subunit catalyzed by elongation factor G. translocation | elongation factor G | cryo-electron microscopy

Recent all-atom crystal structures of 70S ribosome functional complexes provide a detailed description of how the ribosome interacts with its mRNA and tRNA substrates. The structures support the view that the fundamental steps of translation are based on RNA-RNA interactions, which in some cases underwent further refinement as a result of having recruited proteins. The structural basis of the discrimination of cognate tRNA, the high affinity for tRNA in the peptidyl-tRNA site, the structure of the peptidyl transferase catalytic center, the specificity of the exit site for deacylated tRNA and other functional properties of the ribosome are now explained, confirmed or visualized for the first time in complexes containing full-length tRNAs and defined mRNAs. Clues to the structural dynamics of translation are suggested by conformational changes that occur in both tRNA and the ribosome upon complex formation.

New insights into functional flexibility at the peptidyl transferase center (PTC) and its vicinity were obtained by analysis of pleuromutilins binding modes to the ribosome. The crystal structures of Deinococcus radiodurans large ribosomal subunit complexed with each of three pleuromutilin derivatives: retapamulin (SB-275833), SB-280080, and SB-571519, show that all bind to the PTC with their core oriented similarly at the A-site and their C14 extensions pointing toward the P-site. Except for an H-bond network with a single nucleotide, G2061, which involves the essential keto group of all three compounds, only minor hydrophobic contacts are formed between the pleuromutilin C14 extensions and any ribosomal component, consistent with the PTC tolerance to amino acid diversity. Efficient drug binding mode is attained by a mechanism based on induced-fit motions exploiting the ribosomal intrinsic functional flexibility and resulting in conformational rearrangements that seal the pleuromutilin-binding pocket and tightens it up. Comparative studies identified a network of remote interactions around the PTC, indicating that pleuromutilins selectivity is acquired by nonconserved nucleotides residing in the PTC vicinity, in a fashion resembling allosterism. Likewise, pleuromutilin resistant mechanisms involve nucleotides residing in the environs of the binding pocket, consistent with their slow resistance-development rates. antibiotics | functional flexibility | ribosome crystallography | peptidyl transferase center | retapamulin

A computational analysis of codon recognition together with calculated binding free energies using the linear interaction energy method (LIE) in combination with MD simulations for near-cognate and cognate anticodon stem loops is reported. The calculated binding free energies are in excellent agreement with experimental binding constants and reproduce the relative effects of mismatches in the first and second codon position versus a mismatch at the wobble position.

The predictions from molecular dynamics simulations as well as from new crystal structures for the ribosomal peptidyl transfer are analyzed and their implications for the mechanisms of peptidyl transfer and peptidyl-tRNA hydrolysis are examined. The results show that reaction calculations of present simulations can provide an important link between structure and function.

The structural and biochemical studies related to the tmRNA system which performs translational surveillance and ribosome rescue and also some additional roles such as stress management and regulation of transcriptional circuits is presented.

Results show that the ribosome recycling factor binds to the ribosome in a different orientation than the conventional type exhibited by tRNA, indicating that its mechanism of action is not a mimicry of tRNA.

This review evaluates our current understanding of the role played by the ribosomes and their RNA and protein components in the catalysis and regulation of protein synthesis in eukaryotes. The influence of eukaryotic ribosomal RNA processing, chemical modofication, folding, and structure/activities on cellular RNA metabolism is discussed.

The internal transcribed spacer 2 of ribosomal RNA in ticks from the Prostriata lineage shows a 46-49% GC, while those from the Metastriata lineage show 61-62% GC. The secondary structure of the internal transcribed spacer 2 exhibits five domains with stems 1,3,4, and 5 in all the ticks.

This article discusses the maturation and assembly of mitochondrial large subunit and demonstrates that RlmB ortholog is involved in the formation of Gm2251 activity but does not play a role in ribosome assembly or function in Escherichia coli.

A mutation of uracil to cytosine at position 2609 of 23S ribosomal RNA renders Escherichia coli cells resistant to two ketolides while maintaining susceptibility to other macrolides. Mutational and binding data suggest that the macrolide binding site is located in the nascent peptide exit tunnel.

Structural analysis reveal that the large ribosomal subunit of Deinococcus radiodurans eubacterium is composed of intersubunit bridges in unbound subunits, which allow movements of the L1-stalk, facilitating the exit of tRNA.

Electron microscopic analysis reveals that the 80S ribosome of Saccharomyces cerevisiae consists of intersubunit bridges with contacts between the ribosome and the P site-bound peptidyl-tRNA at the molecular level.

Electron microscopic analysis reveals that yeast ribosome-nascent chain containing complexes and associated trimeric Sec61 complex bind to the protein-conducting channel of translating 80S ribosome. A model is proposed for the two functional states of the translating ribosome-channel complex.

E-BP1, which suppresses eIF4E function, is shown to be hypophosphorylated in mitosis. Study results clarify the specific inhibition of cap-dependent translation in mitosis and how eIF4E becomes hypophosphorylated in mitosis. Inhibition of cap-dependent translation in mitosis is caused by phosphorylation modifications that lead to eIF4F complex disruption.

Examination of the path of mRNA through the 70S ribosome reveal that about 30 nucleotides are wrapped in a groove encircling the neck of the 30S subunit. The mRNA entry into the ribosome occurs at about +13 to +15 nucleotide position.

Byline: P. V. Sergiev (1), O. A. Dontsova (1), A. A. Bogdanov (1) Keywords: RNA; ribosome; photoaffinity modification; chemical modification; RNA engineering Abstract: The data on RNA--RNA interactions between the components of the E. colitranslation machinery obtained by X-ray crystallography and chemical methods are compared. The approaches to the study of RNA secondary and tertiary structure are assessed. The following conclusions are made: comparative sequence analysis and compensatory mutations approach both give reliable data on the RNA secondary structure. The chemical modification technique provides good results. Local cleavage of internucleotide bonds by hydroxyl radicals is reliable in the frame of its 40 A resolution, in contrast to the application of copper--phenanthroline complex as a cleavage reagent, which is unreliable. Direct UV irradiation and nitrogen mustard treatment are the best methods of crosslink generation. In vitrotranscription is the only good method for the incorporation of nucleotide analogs in RNA. RNase H hydrolysis and/or nucleotide-specific RNases fingerprints must be applied for the crosslink site determination in parallel with reverse transcription. Author Affiliation: (1) Chemical Faculty, Moscow State University, Moscow, 119899, Russia Article History: Registration Date: 08/10/2004

Eukaryotic mRNA molecules and internal ribosome entry sites (IRESs) in them are discussed in this review article with attention to molecular mechanisms in initiation. Known mechanisms of translation initiation are covered briefly. Use of various mechanisms by specific mRNAs to make translation possible in such situations as apoptosis, mitosis, hypoxia, and some viral infections in which translation of most mRNA is repressed is considered.

The ribosome, the structure of which has recently been characterized at atomic resolution by X-ray crystallography, is discussed in this minireview article. Knowing the structure, which includes knowing morphology of the subunits, gives a major leap in understanding of the mechanism of protein synthesis. Molecular mechanisms of that synthesis are discussed. Phenomena requiring cooperation between the two subunits, such as proofreading of the codon-anticodon interaction, are still not well understood.

Results describe the crystal structures of the large and small subunits of ribosome from Haloarcula marismortui and Thermus thermophilus, respectively, at atomic level resolution. Implications of this new structural information in the understanding of the cellular protein biosynthetic mechanism are discussed.

The ribosome binding kinetics demonstrate that the ribosomes participate actively in aminoacyl-tRNA decoding. Research findings suggest that fully complementary codon-anticodon duplex seem to induce conformational changes leading to acceleration of GTP hydrolysis and peptide bond formation. The model explains the effect of mutations in ribosomal RNA and proteins on the translation fidelity.

Results describe the three-dimensional X-ray crystallographic structures of the small 30S ribosomal subunit of Thermus thermophilus complexed with antibiotics tetracycline, pactamycin, and hygromycin B. Data indicate that the antibiotics bind to different regions of the ribosome subunit and often exhibiting dissimilarities with those of previously determined antibiotic complexes.

Research examines the role of cold shock in reducing the thermal stability of Listeria monocytogenes at the ribosome and fatty acid levels. Results point out that membrane fatty acids are not involved but a 3-hour cold shock at 0 C affected both subunits of the ribosome indicating ribosomal structure instability.

Research describes the crystal structure of the small ribosomal subunit from Thermus thermophilus. The subunit is composed of 19 proteins, a polypeptide of 26 amino acids, and RNA chain of 1457 nucleotides. Data reveal that the subunit exhibits foldings and packing motifs supporting ribosomal architecture.

Researchers used 11.5 Angstrom cryo-electron microscopy to map the E. coli 70S ribosome. This technique allows the ribosome to be visualized without the need for crystallization.

Results demonstrate that the elongation factor G undergoes extensive reorientation with domain 4 at the decoding center of the ribosome with other conformational changes during translocation process as revealed by cryo-electron microscopy.

An electron-density map of the large 50S ribosomal subunit from the bacterium Haloarcular marismortui bacterium has been calculated at 5.0 A resolution. Phases were derived from heavy-atom derivatives, intercrystal density averaging and density-modification methods. Over 300 base pairs of A-form RNA duplex were fitted into the map. A polypeptide exit channel was marked by tungsten cluster compounds bound in a heavy-atom-derivatized crystal.

The structure of the 30S ribosomal subunit is presented, based on an electron-density map at 5.5 A resolution. Double-helical RNA and alpha-helices and Beta sheets of proteins can be identified at this resolution and seven 30S-subunit ribosomal proteins and a large portion of double-stranded RNA helices were fitted into density.

Internal initiation of translation of human rhinovirus (HRV) RNA must have unr, a cellular cytoplasmic RNA-binding protein that has five cold-shock domains. The purification of one of the activities of HeLa cells has been accomplished by using an RNA-affinity column based on the HRV 5' UTR. Two parts were found, one a 38-kD protein, a novel member of the GH-WD repeat protein family having no intrinsic RNA-binding activity and the other a 96-97 kD protein doublet identified as unr. Coimmunoprecipitation with antibodies against either protein shows that they interact with each other.

An ultrastructural study was conducted on ADP-ribosylation factor (ARF)-6 using cryoimmunogold electron microscopy. Results show that ARF-6 is predominantly localized to an intracellular compartment of a cell's pericentriolar region. Results also reveal that the targeted delivery of ARF-6-positive, recycling, endosomal vesicles to plasma membranes requires the activation of ARF.

Newly synthesized polytopic membrane proteins and secretory proteins often share the same target membrane as their primary destination, and in some cases, the cellular machinery that targets and transfers them into or across the membrane. Unlike secretory proteins, which are localized to the external compartment, each polytopic membrane protein molecule must be partitioned among the cytoplasm, the membrane and the external milieu. How does the ribosome-translocon complex cope with the different domains of polytopic membrane proteins?

The importance of the intrinsic ATPase in 80S ribosomes of higher eukaryotes was determined through identification of four binding sites in liver ribosomes of rabbits. Results have shown that the rabbit liver ribosomes contain only three tRNA binding sites A, P and E. The presence of ATP weakened the tight binding of the deacetylated tRNA at the E site. The E site interacted with deacetylated tRNA tightly blocking the binding of aminoacyl-tRNA to the A site.

Structured RNA molecules play essential roles in RNA processing, chromosome maintenance and protein biosynthesis. RNA necessarily uses different strategies than proteins for folding and assembly of complex architectures. The RNA-folding problem is largely an issue of helical packing: how does RNA organize and pack short, double-helical segments to produce active sites and recognition motifs for proteins? Noncanonical base pairs, metal ions and 2[prime]-hydroxyl groups are key elements in RNA higher-order structure formation.

RNase mitochondrial RNA processing enzyme (MRP) is a nucleolar ribonucleoprotein particle that participates in 5.8S ribosomal RNA maturation in eukaryotes. This enzyme shares a polypeptide and an RNA structural motif with ribonuclease P (RNase P), a nuclear endoribo-nuclease originally described in the nucleus that processes tRNA transcripts to generate their mature 5[prime] termini. Both enzymes are also located in mitochondria. This report further characterizes the relationship between RNase MRP and RNase P. Antisense affinity selection with biotinylated 2[prime]-Omethyl oligoribonucleotides and glycerol gradient fractionation experiments demonstrated that small subpopulations of RNase MRP and RNase P associate with each other in vivo in a macromolecular complex, possibly 60-80S preribosomes. This latter notion was supported by fluorescence in situ hybridization experiments with antisense oligonucleotides that localized the RNA components of RNase MRP and RNase P to the nucleolus and to discrete cytoplasmic structures. These findings suggest that small subpopulations of RNase MRP and RNase P are physically associated, and that both may function in ribosomal RNA maturation or ribosome assembly.