Tritium exchange kinetics of yeast ribosomal subunits

Tritium exchange kinetics of 60 S and 40 S ribosomal subunits from Saccharomyces cerevisiae were studied using a rapid centrifugal, ultrafiltration procedure. This assay used commercially available disposable columns and microconcentrators. The tritium-labeled ribosome was separated from the tritiated solvent using a prepacked gel-filtration column. The labeled ribosome was applied to a microconcentrator and the exchange-out kinetics of the ribosome was measured by centrifugation of the ribosome solution and measurement of the amount of radioactivity present in the filtrate. One major advantage of this method is its simplicity and rapidity. With this method, the tritium exchange-out behavior of 60 S and 40 S ribosomal subunits and of subunits during reassociation were determined. The two subunits exhibited different exchange-out rates. Both subunits consisted of multiple classes of exchangeable protons. Considerable conformational changes in both subunits were evident during subunit reassociation, as additions of equal molar quantities of unlabeled 40 S subunits to labeled 60 S subunits caused an immediate increase in the exchange rate. Similarly, an increase in the exchange rate in the small subunits upon addition of unlabeled 60 S subunits was observed.     

Translational accuracy of a tethered ribosome

Ribosomes are evolutionary conserved ribonucleoprotein complexes that function as two separate subunits in all kingdoms. During translation initiation, the two subunits assemble to form the mature ribosome, which is responsible for translating the messenger RNA. When the ribosome reaches a stop codon, release factors promote translation termination and peptide release, and recycling factors then dissociate the two subunits, ready for use in a new round of translation. A tethered ribosome, called Ribo-T, in which the two subunits are covalently linked to form a single entity, was recently described in Escherichia coli. A hybrid ribosomal RNA (rRNA) consisting of both the small and large subunit rRNA sequences was engineered. The ribosome with inseparable subunits generated in this way was shown to be functional and to sustain cell growth. Here, we investigated the translational properties of Ribo-T. We analyzed its behavior during amino acid misincorporation, −1 or +1 frameshifting, stop codon readthrough, and internal translation initiation. Our data indicate that covalent attachment of the two subunits modifies the properties of the ribosome, altering its ability to initiate and terminate translation correctly.     

Definition of bases in 23S rRNA essential for ribosomal subunit association

The ribosome is a two-subunit molecular machine, sporting a working cycle that involves coordinated movements of the subunits. Recent structural studies of the 70S ribosome describe a rather large number of intersubunit contacts, some of which are dynamic during translocation. We set out to determine which intersubunit contacts are functionally indispensable for the association of ribosome subunits by using a modification interference approach. Modification of the N-1 position of A715, A1912, or A1918 in Escherichia coli 50S subunits is strongly detrimental to 70S ribosome formation. This result points to 23S rRNA helices 34 and 69, and thus bridges B2a and B4, as essential for ensuring stability of the 70S ribosome.     

Thiostrepton, an Inhibitor of 50S Ribosome Subunit Function

Thiostrepton inhibits (14)C-leucine incorporation by intact cells of Bacillus megaterium as well as (14)C-phenylalanine incorporation by a poly U-directed extract of Escherichia coli. Extracts of E. coli which are pretreated by incubation with thiostrepton cannot be reactivated by dialysis to more than 5% of their former activity. The 50S ribosome subunit appears to be the site of thiostrepton action, since protein-synthesizing activity can be restored to dialyzed pretreated extracts by supplementation with 50S ribosome subunits but not with 30S ribosome subunits. This technique also provides a simple sensitive method for detection of the biological activity of very small amounts of 50S ribosome subunits.     

Dom34:hbs1 plays a general role in quality-control systems by dissociation of a stalled ribosome at the 3' end of aberrant mRNA

Translation arrest leads to an endonucleolytic cleavage of mRNA that is termed no-go decay (NGD). It has been reported that the Dom34:Hbs1 complex stimulates this endonucleolytic cleavage of mRNA induced by translation arrest in vivo and dissociates subunits of a stalled ribosome in vitro. Here we report that Dom34:Hbs1 dissociates the subunits of a ribosome that is stalled at the 3' end of mRNA in vivo, and has a crucial role in both NGD and nonstop decay. Dom34:Hbs1-mediated dissociation of a ribosome that is stalled at the 3' end of mRNA is required for degradation of a 5'-NGD intermediate. Dom34:Hbs1 facilitates the decay of nonstop mRNAs from the 3' end by exosomes and is required for the complete degradation of nonstop mRNA decay intermediates. We propose that Dom34:Hbs1 stimulates degradation of the 5'-NGD intermediate and of nonstop mRNA by dissociating the ribosome that is stalled at the 3' end of the mRNA.     

A novel GTPase activated by the small subunit of ribosome

The GTPase activity of Escherichia coli YjeQ, here named RsgA (ribosome small subunit-dependent GTPase A), has been shown to be significantly enhanced by ribosome or its small subunit. The enhancement of GTPase activity was inhibited by several aminoglycosides bound at the A site of the small subunit, but not by a P site-specific antibiotic. RsgA stably bound the small subunit in the presence of GDPNP, but not in the presence of GTP or GDP, to dissociate ribosome into subunits. Disruption of the gene for RsgA from the genome affected the growth of the cells, which predominantly contained the dissociated subunits having only a weak activation activity of RsgA. We also found that 17S RNA, a putative precursor of 16S rRNA, was contained in the small subunit of the ribosome from the RsgA-deletion strain. RsgA is a novel GTPase that might provide a new insight into the function of ribosome.     

Mutations in the intersubunit bridge regions of 23 S rRNA

The large and small subunits of the ribosome are joined by a series of bridges that are conserved among mitochondrial, bacterial, and eukaryal ribosomes. In addition to joining the subunits together at the initiation of protein synthesis, a variety of other roles have been proposed for these bridges. These roles include transmission of signals between the functional centers of the two subunits, modulation of tRNA-ribosome and factor-ribosome interactions, and mediation of the relative movement of large and small ribosomal subunits during translocation. The majority of the bridges involve RNA-RNA interactions, and to gain insight into their function, we constructed mutations in the 23 S rRNA regions involved in forming 7 of the 12 intersubunit bridges in the Escherichia coli ribosome. The majority of the mutants were viable in strains expressing mutant rRNA exclusively but had distinct growth phenotypes, particularly at 30 degrees C, and the mutant ribosomes promoted a variety of miscoding errors. Analysis of subunit association activities both in vitro and in vivo indicated that, with the exception of the bridge B5 mutants, at least one mutation at each bridge site affected 70 S ribosome formation. These results confirm the structural data linking bridges with subunit-subunit interactions and, together with the effects on decoding fidelity, indicate that intersubunit bridges function at multiple stages of protein synthesis.     

The ribosome as a drug target

The elucidation of the crystal structure of the ribosome and its subunits has dramatically increased our understanding of this organelle and the molecular interactions that determine its functional capabilities. Two recent publications, one on the structure of the bacterial ribosome at 3.5A resolution and one on the identification of functionally relevant sites within the small subunit rRNA, illustrate the importance of interdisciplinary approaches in exploiting the ribosome as a drug target.     

Observations of self-aggregation and dissociation of E. coli ribosomes by optical mixing spectroscopy

Measurements of the diffusion coefficient of E. coli ribosomes by the technique of optical mixing spectroscopy are used to show that these ribosomes, when incubated in a 4°C cold room, undergo a slow aggregation with time. Even in the presence of this aggregation, it was possible to observe the diffusive character of the single 70s ribosome. It was found that at high (≥0.100 molar) KCl concentrations the ribosomes broke into their subunits. At moderate KCl concentrations they were relatively stable and at a very low KCl concentration (0.005 M) they were observed to dimerize. The values of the diffusion coefficients of the 50s subunits, the 70s single ribosome and the ribosome dimer determined in these measurements are consistent with currently accepted values.     

Relationship between synthesis of ribosomes and RNA polymerase in Bacillus subtilis

The relationship between RNA polymerase synthesis and ribosome synthesis has been investigated in Bacillus subtilis growing at different rates. The amount of polymerase was determined by quantitation of polymerase subunits on polyacrylamide gels, while ribosome synthesis was estimated from accumulation of total RNA. The ratio of ribosome to RNA polymerase synthesis was 5:1 at slower growth rates and increased with growth rate. Bacillus subtilis was estimated to contain between 10-and 2-fold excess of RNA polymerase depending on its growth rate.     

Mechanism of action of the wheat germ ribosome dissociation factor: Interaction with the 60 S subunit

Recently a ribosome dissociation factor that stimulates natural mRNA translation has been isolated from extracts of wheat germ. In this investigation, we have studied the subunit site of action of the purified ribosome dissociation factor (eucaryotic initiation), eIF-6. The following evidence strongly indicates that eIF-6 acts as a dissociation factor by binding to the 60 S ribosomal subunit and preventing its interaction with the 40 S subunit. Incubation of 60 S subunits with eIF-6 reduces the formation of 80 S monosomes when 40 S subunits are subsequently added at 5 mm Mg²⁺. The 40 S subunits preincubated with eIF-6 reassociate normally with 60 S subunits. ¹⁴C-labeled eIF-6 binds to 60 S subunits but not to 40 S subunits. Slight binding to 80 S ribosomes is also observed. The interaction of eIF-6 with the 60 S subunit requires an elevated temperature, and occurs rapidly at 37 °C.     

Era and RbfA have overlapping function in ribosome biogenesis in Escherichia coli

A cold-shock protein, RbfA (ribosome-binding factor A), is essential for cell growth at low temperature. In an rbfA-deletion strain, 30S and 50S ribosomal subunits increase relative to 70S monosomes with concomitant accumulation of a precursor 16S rRNA (17S rRNA). Recently, we have reported that overexpression of Era, an essential GTP-binding protein, suppresses not only the cold-sensitive cell growth but also defective ribosome biogenesis in the rbfA-deletion strain. Here, in order to elucidate how RbfA and Era functionally overlap, we characterized a cold-sensitive Era mutant (a point mutation at the Glu-200 to Lys; E200K) which shows a similar phenotype as the rbfA-deletion strain; accumulation of free ribosome subunits and 17S rRNA. To examine the effect of E200K in the rbfA-deletion strain, we constructed an E200K-inducible expression system. Interestingly, unlike wild-type Era, overexpression of Era(E200K) protein in the rbfA-deletion strain severely inhibited cell growth even at permissive temperature with further concomitant reduction of 16S rRNA. Purified Era(E200K) protein binds to 30S ribosomal subunits in a nucleotide-dependent manner like wild-type Era and retains both GTPase and autophosphorylation activities. Furthermore, we isolated spontaneous revertants of the E200K mutant. These revertants partially suppressed the accumulation of 17S rRNA. All the spontaneous mutations were found to result in higher Era(E200K) expression. These results suggest that the Era(E200K) protein has an impaired function in ribosome biogenesis without losing its ribosome binding activity. The severe growth defect caused by E200K in the rbfA-deletion strain may be due to competition between intrinsic wild-type Era and overexpressed Era(E200K) for binding to 30S ribosomal subunits. We propose that Era and RbfA have an overlapping function that is essential for ribosome biogenesis, and that RbfA becomes dispensable only at high temperatures because Era can complement its function only at higher temperatures.     

Initiation of ribosome degradation during starvation in Escherichia coli

Ribosomes are known to be degraded under conditions of nutrient limitation. However, the mechanism by which a normally stable ribosome becomes a substrate for the degradation machinery has remained elusive. Here, we present in vitro and in vivo data demonstrating that free ribosome subunits are the actual targets of the degradative enzymes, whereas 70S particles are protected from such degradation. Conditions that increase the formation of subunits both in vitro and in vivo lead to enhanced degradation, while conditions favoring the presence of intact 70S ribosomes prevent or reduce breakdown. Thus, the simple formation of free 50S and 30S subunits is sufficient to serve as the initiation mechanism that allows endoribonuclease cleavage and subsequent ribosome breakdown.     

In vitro protein folding by E. coli ribosome: unfolded protein splitting 70S to interact with 50S subunit

Folding of unfolded protein on Escherichia coli 70S ribosome is accompanied by rapid dissociation of the ribosome into 50S and 30S subunits. The dissociation rate of 70S ribosome with unfolded protein is much faster than that caused by combined effect of translation and polypeptide release factors known to be involved in the dissociation of ribosome into subunits. The protein then reaches a “folding competent” state on 50S and is released to take up native conformation by itself. Release before attaining the folding competent state or prevention of release by cross-linking it with ribosome, would not allow the protein to get back to its native conformation.     

Chain initiation factor 3 crosslinks to E. coli 30S and 50S ribosomal subunits and alters the UV absorbance spectrum of 70S ribosomes

We report a direct procedure to determine the proteins near the IF-3 binding site in purified 30S and 50S ribosomal subunits. This procedure introduces only limited numbers of cleavable crosslinks between IF-3 and its nearest neighbors. The cleavable crosslinking reagent, 2-iminothiolane, was used to crosslink IF-3 in place to both 30S and 50S subunits. Ribosomal proteins S9/S11, S12, L2, L5 and L17 were found, by this approach, to be in close proximity to the factor in purified IF-3-subunit complexes. In addition, IF-3 was shown to alter the ultraviolet absorbance spectrum of E. coli 70S ribosomes at 10 mM Mg2+. The magnitude of the observed difference spectrum at a constant IF-3/ribosome ratio of 1.0, is linearly dependent upon ribosome concentration over the range 5 nM - 55 nM. Titration experiments indicated that the observed effect is maximal at an IF-3/ribosome ratio of approximately 1.0. These results are taken to indicate a conformational change in the 70S ribosome induced by IF-3.     

Exploration of RNA 3' terminal locations in rat ribosome.

The locations of the 3' ends of RNAs in rat ribosome were studied by a fluorescence-labeling method combined with high hydrostatic pressure and agarose electrophoresis. Under physiological conditions, only the 3' ends of 28 S and 5.8 S RNA in 80 S ribosome could be labeled with a high sensitive fluorescent probe - fluorescein 5-thiosemicarbazide (FTSC), indicating that the 3' termini of 28 S and 5.8 S RNA were located on or near the surface of 80 S ribosome. The 3' terminus of 5 S RNA could be attacked by FTSC only in the case of the dissociation of the 80 S ribosome into two subunits induced by high salt concentration (1 M KCl) or at high hydrostatic pressure, showing that the 3' end of 5 S RNA was located on the interface of two subunits. However, no fluorescence-labeled 18 S RNA could be detected under all the conditions studied, suggesting that the 3' end of 18 S RNA was either located deeply inside ribosome or on the surface but protected by proteins. It was interesting to note that modifications of the 3' ends of ribosomal RNAs including oxidation with NaIO4, reduction with KBH4 and labeling with fluorescent probe did not destroy the translation activity of ribosome, indicating that the 3' ends of RNAs were not involved in the translation activity of ribosome.     

Structural changes of ribosome by the action of ethylene glycol.

The denaturation of ribosome and RNA by ethylene glycol (EG) has been studied in an attempt to further understand the conformation and stability of the ribosome. At high concentrations of EG, the ribosome, its subunits, and 16S RNA undergo drastic structural changes as shown by circular dichroism, ultraviolet absorption spectroscopy, and sedimentation velocity. Two separate conformational transitions were observed for the 30S subunit; one from 30 to 50% EG and another from 60 to 90% EG. This observation suggests the presence of two "domains" in the 30S subunit which differ in their stability. However, the 50S subunit undergoes a single sharp transition at 60 to 90% EG, consistent with the notion of a highly cooperative conformation. Association of the subunits stablizes part of the 30S subunit since the transition curve for the 70S ribosome does not exhibit significant change at the low EG concentration region as seen for the 30S subunit. Removal of proteins from the 30S subunit broadens the transition curve to lower EG concentrations and suggests the role of proteins in stabilizing the conformation of the 16S RNA.     

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 pre-ribosomal network

Recent achievements in yeast functional proteomics have significantly advanced our knowledge about ribosome biogenesis. Here, we present a program developed to integrate data from various proteome analyses with cell biological data on components present in the ribosome producing factories. This program allows users to attribute factors to certain complexes and to specific steps of ribosome biogenesis. Thus, it helps to gain novel insights into the complex network involved in maturation of ribosomal subunits. The database can be accessed at the URL http://www.pre-ribosome.de.     

Specific visualization of ribosomal RNA in the intact ribosome by electron spectroscopic imaging

The recently developed electron microscopic technique of electron spectroscopic imaging has been used to map the distribution of phosphorus, and therefore of RNA, in situ in the ribosomal subunits of E. coli. The results indicate that the RNA moiety of both subunits is concentrated toward the centre of the particle somewhat more than is the total mass, but reaches the outer surface at several places. The micrographs also reveal certain distinctive features in the shape of the RNA component that may be related to the overall shape of the ribosome. The method yielded a reasonably accurate estimate of the phosphorus content of the 30 S ribosome.