Structural Insights into ribosome recycling factor interactions with the 70S ribosome

At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. Upon binding of either E. coli or Thermus thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix 69 in the large subunit moves away from the small subunit toward RRF by 8 Å, thereby disrupting a key contact between the small and large ribosomal subunits termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of helix 69 involves an ordered-to-disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling.     

The role of ribosome recycling factor in dissociation of 70S ribosomes into subunits

Protein synthesis is initiated on ribosomal subunits. However, it is not known how 70S ribosomes are dissociated into small and large subunits. Here we show that 70S ribosomes, as well as the model post-termination complexes, are dissociated into stable subunits by cooperative action of three translation factors: ribosome recycling factor (RRF), elongation factor G (EF-G), and initiation factor 3 (IF3). The subunit dissociation is stable enough to be detected by conventional sucrose density gradient centrifugation (SDGC). GTP, but not nonhydrolyzable GTP analog, is essential in this process. We found that RRF and EF-G alone transiently dissociate 70S ribosomes. However, the transient dissociation cannot be detected by SDGC. IF3 stabilizes the dissociation by binding to the transiently formed 30S subunits, preventing re-association back to 70S ribosomes. The three-factor-dependent stable dissociation of ribosomes into subunits completes the ribosome cycle and the resulting subunits are ready for the next round of translation.     

Comparison of fungal 80 S ribosomes by cryo-EM reveals diversity in structure and conformation of rRNA expansion segments

Compared to the prokaryotic 70 S ribosome, the eukaryotic 80 S ribosome contains additional ribosomal proteins and extra segments of rRNA, referred to as rRNA expansion segments (ES). These eukaryotic-specific rRNA ES are mainly on the periphery of the 80 S ribosome, as revealed by cryo-electron microscopy (cryo-EM) studies, but their precise function is not known. To address the question of whether the rRNA ES are structurally conserved among 80 S ribosomes of different fungi we performed cryo-electron microscopy on 80 S ribosomes from the thermophilic fungus Thermomyces lanuginosus and compared it to the Saccharomyces cerevisiae 80 S ribosome. Our analysis reveals general structural conservation of the rRNA expansion segments but also changes in ES27 and ES7/39, as well as the absence of a tertiary interaction between ES3 and ES6 in T. lanuginosus. The differences provide a hint on the role of rRNA ES in regulating translation. Furthermore, we show that the stalk region and interactions with elongation factor 2 (eEF2) are different in T. lanuginosus, exhibiting a more extensive contact with domain I of eEF2.     

Structure of the Bacillus subtilis hibernating 100S ribosome reveals the basis for 70S dimerization

Under stress conditions, such as nutrient deprivation, bacteria enter into a hibernation stage, which is characterized by the appearance of 100S ribosomal particles. In Escherichia coli, dimerization of 70S ribosomes into 100S requires the action of the ribosome modulation factor (RMF) and the hibernation‐promoting factor (HPF). Most other bacteria lack RMF and instead contain a long form HPF (LHPF), which is necessary and sufficient for 100S formation. While some structural information exists as to how RMF and HPF mediate formation of E. coli 100S (Ec100S), structural insight into 100S formation by LHPF has so far been lacking. Here we present a cryo‐EM structure of the Bacillus subtilis hibernating 100S (Bs100S), revealing that the C‐terminal domain (CTD) of the LHPF occupies a site on the 30S platform distinct from RMF. Moreover, unlike RMF, the BsHPF‐CTD is directly involved in forming the dimer interface, thereby illustrating the divergent mechanisms by which 100S formation is mediated in the majority of bacteria that contain LHPF, compared to some γ‐proteobacteria, such as E. coli.     

Structural study of translating 70 S ribosomes from Escherichia coli: I. Electron microscopy

Translating 70 S ribosomes of Escherichia coli either in the pre-translocation or in the post-translocation state have been prepared by using the cell-free translation system in poly(U)—S—S—Sepharose columns [Methods Enzymol. (1979) 59, 382–398]. Electron microscopy study of the preparations has demonstrated that: (1) the mutual orientation of the ribosomal subunits in the translating ribosomes is the same as proposed by Lake for routine 30 S·50 S couples [J. Mol. Biol. (1976) 105, 111–130]; (2) the L7/L12 stalk of the 50 S subunit sticks out from the 70 S particle and does not join the 30 S subunit; (3) pre-translocation and post-translocation state ribosomes do not differ in mutual orientation of the subunits and in the position of the L7/L12 stalk, within the limits of electron microscopy resolution.     

Native 3D structure of eukaryotic 80s ribosome: morphological homology with E. coli 70S ribosome

A three-dimensional reconstruction of the eukaryotic 80S monosome from a frozen-hydrated electron microscopic preparation reveals the native structure of this macromolecular complex. The new structure, at 38A resolution, shows a marked resemblance to the structure determined for the E. coli 70S ribosome (Frank, J., A. Verschoor, Y. Li, J. Zhu, R.K. Lata, M. Radermacher, P. Penczek, R. Grassucci, R.K. Agrawal, and Srivastava. 1996b. In press; Frank, J., J. Zhu, P. Penczek, Y. Li, S. Srivastava ., A. Verschoor, M. Radermacher, R. Grassucci, R.K. Lata, and R. Agrawal. 1995. Nature (Lond.).376:441-444.) limited to a comparable resolution, but with a number of eukaryotic elaborations superimposed. Although considerably greater size and intricacy of the features is seen in the morphology of the large subunit (60S vs 50S), the most striking differences are in the small subunit morphology (40S vs 30S): the extended beak and crest features of the head, the back lobes, and the feet. However, the structure underlying these extra features appears to be remarkably similar in form to the 30S portion of the 70S structure. The intersubunit space also appears to be strongly conserved, as might be expected from the degree of functional conservation of the ribosome among kingdoms (Eukarya, Eubacteria, and Archaea). The internal organization of the 80S structure appears as an armature or core of high-density material for each subunit, with the two cores linked by a single bridge between the platform region of the 40S subunit and the region below the presumed peptidyltransferase center of the 60S subunit. This may be equated with a close contact of the 18S and 28S rRNAs in the translational domain centered on the upper subunit:subunit interface.     

Intralineage diversity of archaebacterial ribosomes: A dichotomy of ribosome features separates sulfur-dependent archaebacteria and methanococcaceae from other archaebacterial taxa

Summary The aggregate masses and relative protein contents of eubacterial and archaebacterial ribosomes have been estimated from the buoyant densities of the ribosomal subunits and the anhydrous weights of the rRNA species. In contrast to the situation in eubacteria, archaebacterial ribosomes fall into two size classes that differ only in the relative abundances of their protein moieties. One class comprises eubacterial-sized particles (2.3-megadalton (Mdal) monomer; 1.5-Mdal and 0.8-Mdal large and small subunits, respectively) having a eubacterial composition of roughly one-third protein and two-thirds RNA. The other class comprises ribosomes heavier than those of eubacteria (3-Mdal monomer; 1.8-Mdal and 1.2-Mdal subunits) and having the same protein/RNA ratio as eukaryotic ribosomes (55% and 50% protein for the small and large subunits, respectively). Eubacterialsized ribosomes are harbored by extreme halophiles and all methanogens but the Methanococcaceae. Ribosomes heavier than those of eubacteria are found in the Methanococcaceae and all sulfur-dependent thermophiles. The data indicate that a change in ribosome structure occurred within the methanogen branch; therefore, although ribosome composition is distributed in archaebacteria, its distribution does not break them into two separate kingdoms: The Methanococcaceae and Methanobacteriaceae are related to each other far more closely than either is to the sulfur-dependent thermophiles, and the root of the archaebacterial tree definitely does not lie betweenMethanobacterium andMethanococcus. We surmise that ribosomes larger than those of eubacteria represent a more rudimentary organelle structure that became fixed owing to nonparallel evolution of the translational machinery in archaebacteria.     

Structural insights into the interplay of protein biogenesis factors with the 70S ribosome

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Streptomycin-induced conformational changes in the 70-S bacterial ribosome.

Conformational alterations induced by streptomycin in the bacterial ribosome have been investigated using as probes, ethidium bromide, N-[14C]ethylmaleimide and a spin label nitroxide analog of N-ethylmaleimide. 1. The binding of the antibiotic to the ribosome does not affect the reactivity of sulfhydryl groups towards N-ethylmaleimide. 2. The motional freedom of spin labels bound to ribosomal proteins S1 and S18 is increased but it is hardly affected at other labeled sites. This observation suggests that the binding of streptomycin causes a local loosening of the ribosomal structure. 3. Ribosomes are found to bind less ethidium bromide in the presence of streptomycin, which suggests that the binding of streptomycin decreases the degree of organization of ribosomal RNA.     

34 Cyo-EM visualization of Mycobacterium 70S ribosome reveals unique structural components at the function sites

The 3D structures of prokaryotic and eukaryotic ribosomes by crystallography and electron microscopy have revealed that they share an evolutionarily conserved core (Schmeing & Ramakrishnan, 2009), but each of the ribosomes contains its own set of specific proteins (or extensions of conserved proteins) and expansion segments of rRNAs (Melnikov et al., 2012). How these differences correlate to function still remains largely unknown. A 3D cryo-EM map of the 70S ribosome from Mycobacterium smegmatis (Msm70S) unveiled striking new structural features (Shasmal & Sengupta, 2012). The core of the Msm70S shows overall similarity with the core of the Escherichia coli 70S ribosome while containing additional mass in the periphery and solvent exposed sides. Some of the Mycobacterium ribosomal proteins are significantly bigger as compared to the E. coli counter parts. The rRNAs also contain extra helices, also revealed by their secondary structures. Most of the additional density of the Msm70S can be largely attributed to the extra helices present in the rRNAs, and extra domains of homologous proteins. One of the most notable features appears in the large subunit near L1 stalk as a structure forming a long helix with its upper end located in the vicinity of the mRNA exit channel (which we term the ‘steeple’). We propose that the prominent helical structure in mycobacterium 23S rRNA participates in modulating different steps of translation, especially the E site tRNA exit mechanism and propagation of mRNA 5′ end.     

Structural dynamics of bacterial ribosomes: IV. Classification of ribosomes by subunit interaction

Previous studies in this series (M. Noll et al., 1973a,b; Noll & Noll, 1974) have established that in Escherichia coli the ability of subunits to form vacant 70 S ribosome couples at 10 mm-Mg²⁺ is a stringent condition for activity in the translation of natural messenger (R17 RNA). The present study examines the structural basis of subunit interaction. It is found that vacant ribosome couples prepared by various methods fall into two classes, “tight” couples and “loose” couples, that differ in the affinity of their subunits for each other. Detection and separation of the two particle species is possible by ultracentrifugation. When analyzed on sucrose gradients at 6 mm-Mg²⁺ and moderate speed (30,000 revs/min), tight couples sediment as undissociated 70 S ribosomes, whereas loose couples are completely dissociated and sediment as 30 S and 50 S subunits. At 15 mm-Mg²⁺ in the gradient, both species sediment as a 70S peak. At 10 mm-Mg²⁺ and 60,000 revs/min, two peaks (63 S and 55 S) are seen because the high hydrostatic pressure causes more pronounced dissociation of the loose than of the tight couples.Association is dependent on the state of each subunit. Removal of Mg²⁺ produces 30 S b-particles that are unable to associate with 50 S subunits unless reconverted to the 30 S a-form by thermal activation according to Zamir et al. (1971). In the dissociated state, 50 S subunits tend to change irreversibly to a 50 S b-modification that produces loose couples upon association with 30 S a-subunits. The 50 S a → 50 S b transition could not be related to breaks in 23 S RNA detectable by sedimentation analysis. However, mild treatment of 50 S a-subunits with RNase produces particles that associate with 30 S a-subunits to couples that are less stable than the loose couples resulting from a dissociation/association step.Fresh S-30 extracts contain only tight couples (approx. 80%) and subunits (approx. 20%). Our results suggest that loose couples are artefacts derived from tight couples by a structural or conformational modification.Interaction-free subunits that previously were found to form a primitive initiation complex with poly(U) and tRNA_Phe (Schreier & Noll, 1970,1971), and to be active in phenylalanine polymerization, are shown to consist of the b-form of each subunit.It is likely that conflicting results obtained in the study of the mechanism of initiation and other aspects of ribosome function are due to the lack of structural criteria required for standardizing the ribosome preparation used by different investigators. This study provides simple methods and criteria to classify and separate physically all ribosome and ribosome subunits that have been observed into well-defined classes of predictable activity.     

The complete structure of the chloroplast 70S ribosome in complex with translation factor pY

Chloroplasts are cellular organelles of plants and algae that are responsible for energy conversion and carbon fixation by the photosynthetic reaction. As a consequence of their endosymbiotic origin, they still contain their own genome and the machinery for protein biosynthesis. Here, we present the atomic structure of the chloroplast 70S ribosome prepared from spinach leaves and resolved by cryo‐EM at 3.4 Å resolution. The complete structure reveals the features of the 4.5S rRNA, which probably evolved by the fragmentation of the 23S rRNA, and all five plastid‐specific ribosomal proteins. These proteins, required for proper assembly and function of the chloroplast translation machinery, bind and stabilize rRNA including regions that only exist in the chloroplast ribosome. Furthermore, the structure reveals plastid‐specific extensions of ribosomal proteins that extensively remodel the mRNA entry and exit site on the small subunit as well as the polypeptide tunnel exit and the putative binding site of the signal recognition particle on the large subunit. The translation factor pY, involved in light‐ and temperature‐dependent control of protein synthesis, is bound to the mRNA channel of the small subunit and interacts with 16S rRNA nucleotides at the A‐site and P‐site, where it protects the decoding centre and inhibits translation by preventing tRNA binding. The small subunit is locked by pY in a non‐rotated state, in which the intersubunit bridges to the large subunit are stabilized.     

Solution structure of the E. coli 70S ribosome at 11.5 A resolution

Over 73,000 projections of the E. coli ribosome bound with formyl-methionyl initiator tRNAf(Met) were used to obtain an 11.5 A cryo-electron microscopy map of the complex. This map allows identification of RNA helices, peripheral proteins, and intersubunit bridges. Comparison of double-stranded RNA regions and positions of proteins identified in both cryo-EM and X-ray maps indicates good overall agreement but points to rearrangements of ribosomal components required for the subunit association. Fitting of known components of the 50S stalk base region into the map defines the architecture of the GTPase-associated center and reveals a major change in the orientation of the alpha-sarcin-ricin loop. Analysis of the bridging connections between the subunits provides insight into the dynamic signaling mechanism between the ribosomal subunits.     

Composition and structure of the 80S ribosome from the green alga Chlamydomonas reinhardtii: 80S ribosomes are conserved in plants and animals

We have conducted a proteomic analysis of the 80S cytosolic ribosome from the eukaryotic green alga Chlamydomonas reinhardtii, and accompany this with a cryo-electron microscopy structure of the ribosome. Proteins homologous to all but one rat 40S subunit protein, including a homolog of RACK1, and all but three rat 60S subunit proteins were identified as components of the C. reinhardtii ribosome. Expressed Sequence Tag (EST) evidence and annotation of the completed C. reinhardtii genome identified genes for each of the four proteins not identified by proteomic analysis, showing that algae potentially have a complete set of orthologs to mammalian 80S ribosomal proteins. Presented at 25A, the algal 80S ribosome is very similar in structure to the yeast 80S ribosome, with only minor distinguishable differences. These data show that, although separated by billions of years of evolution, cytosolic ribosomes from photosynthetic organisms are highly conserved with their yeast and animal counterparts.     

Release of 70 S ribosomes from polysomes in Escherichia coli

In order to determine whether ribosomes are released from messenger RNA as intact particles or as subunits, polysomes of Escherichia coli labeled with heavy isotopes were allowed to run off together with “light” polysomes. The normally rapid post-run-off exchange of subunits by free ribosomes was virtually eliminated by two means: the use of purified polysomes (relatively free of initiation factors), and incubation at a lower temperature (25 °C), or at a somewhat higher Mg²⁺ concentration (12 to 14 mm), than is conventional. Under these conditions ribosomes released by run-off or by puromycin accumulated without subunit exchange. Hence, even though the ribosome normally initiates via subunits, it is released from RNA by a conformational change in the intact 70 S particle, rather than by dissociation.     

A-site residues move independently from P-site residues in all-atom molecular dynamics simulations of the 70S bacterial ribosome

The ribosome is a large macromolecular machine, and correlated motion between residues is necessary for coordinating function across multiple protein and RNA chains. We ran two all-atom, explicit solvent molecular dynamics simulations of the bacterial ribosome and calculated correlated motion between residue pairs by using mutual information. Because of the short timescales of our simulation (ns), we expect that dynamics are largely local fluctuations around the crystal structure. We hypothesize that residues that show coupled dynamics are functionally related, even on longer timescales. We validate our model by showing that crystallographic B-factors correlate well with the entropy calculated as part of our mutual information calculations. We reveal that A-site residues move relatively independently from P-site residues, effectively insulating A-site functions from P-site functions during translation.     

CysG structure reveals tetrapyrrole-binding features and novel regulation of siroheme biosynthesis

Sulfur metabolism depends on the iron-containing porphinoid siroheme. In Salmonella enterica, the S-adenosyl-L-methionine (SAM)-dependent bismethyltransferase, dehydrogenase and ferrochelatase, CysG, synthesizes siroheme from uroporphyrinogen III (uro'gen III). The reactions mediated by CysG encompass two branchpoint intermediates in tetrapyrrole biosynthesis, diverting flux first from protoporphyrin IX biosynthesis and then from cobalamin (vitamin B(12)) biosynthesis. We determined the first structure of this multifunctional siroheme synthase by X-ray crystallography. CysG is a homodimeric gene fusion product containing two structurally independent modules: a bismethyltransferase and a dual-function dehydrogenase-chelatase. The methyltransferase active site is a deep groove with a hydrophobic patch surrounded by hydrogen bond donors. This asymmetric arrangement of amino acids may be important in directing substrate binding. Notably, our structure shows that CysG is a phosphoprotein. From mutational analysis of the post-translationally modified serine, we suggest a conserved role for phosphorylation in inhibiting dehydrogenase activity and modulating metabolic flux between siroheme and cobalamin pathways.