Small protein B interacts with the large and the small subunits of a stalled ribosome during trans-translation

During trans-translation, stalled bacterial ribosomes are rescued by small protein B (SmpB) and by transfer-messenger RNA (tmRNA). Stalled ribosomes switch translation from the defective messages to a short internal reading frame on tmRNA that tags the nascent peptide chain for degradation and recycles the ribosomes. We present evidences that SmpB binds the large and small ribosomal subunits in vivo and in vitro. The binding between SmpB and the ribosomal subunits is very tight, with a dissociation constant of 1.7 × 10⁻¹⁰ M, similar to its K_D for the 70S ribosome or for tmRNA. tmRNA displaces SmpB from its 50S binding but not from the 30S. In vivo, SmpB is detected on the 50S when trans-translation is impaired by lacking tmRNA or a functional SmpB. SmpB contacts the large subunit transiently and early during the trans-translational process. The affinity of SmpB for the two ribosomal subunits is modulated by tmRNA in the course of trans-translation. It is the first example of two copies of the same protein interacting with two different functional sites of the ribosomes.     

Maize mitochondria: purification and characterization of ribosomes and ribosomal ribonucleic Acid

Mitochondria were prepared from etiolated maize shoots (Zea mays L. var. McNair 508) by homogenization followed by differential centrifugation and equilibrium banding in discontinuous sucrose or Renografin-sucrose gradients. Mitochondria prepared by sucrose banding showed better physiological integrity than those prepared by renografin-sucrose banding, although both procedures yielded mitochondria that showed respiratory control and coupling of oxidation to phosphorylation of ADP. Mitochondria prepared by Renografin-sucrose banding were free of dectectable cytoplasmic ribosomal RNA, while sucrose banding resulted in a low level of contamination. Ribosomes isolated from mitochondria sedimented at about 78S, with subunits sedimenting at 60 and 44S. Using Escherichia coli ribosomal RNA as internal standards, the molecular weights of mitochondrial ribosomal RNAs were found to be 0.74 to 0.75 and 1.26 × 10⁶ daltons by polyacrylamide gel electrophoresis, before or after denaturation in formaldehyde. Cytoplasmic ribosomal RNA molecular weights were 0.70 and 1.26 × 16⁶ before denaturation, and 0.68 and 1.5 × 10⁶ after denaturation, suggesting an unusual reaction of the heavy ribosomal RNA to formaldehyde.     

Surface topography of the Bacillus stearothermophilus ribosome.

Summary The surface topography of the intact 70S ribosome and free 30S and 50S subunits from Bacillus stearothermophilus strain 2184 was investigated by lactoperoxidase-catalyzed iodination. Two-dimensional polyacrylamide gel electrophoresis was employed to separate ribosomal proteins for analysis of their reactivity. Free 50S subunits incorporated about 18% more ¹²⁵I than did 50S subunits derived from 70S ribosomes, whereas free 30S subunits and 30S subunits derived from 70S ribosomes incorporated similar amounts of ¹²⁵I. Iodinated 70S ribosomes and subunits retained 62–78% of the protein synthesis activity of untreated particles and sedimentation profiles showed no gross conformational changes due to iodination. The proteins most reactive to enzymatic iodination were S4, S7, S10 and Sa of the small subunit and L2, L4, L5/9, L6 and L36 of the large subunit. Proteins S2, S3, S7, S13, Sa, L5/9, L10, L11 and L24/25 were labeled substantially more in the free subunits than in the 70S ribosome. Other proteins, including S5, S9, S12, S15/16, S18 and L36 were more extensively iodinated in the 70S ribosome than in the free subunits. The locations of tyrosine residues in some homologus ribosomal proteins from B. stearothermophilus and E. coli are compared.     

Isolation and characterization of cytoplasmic and chloroplastic ribosomes and their ribosomal RNAs from the diatom Cylindrotheca fusiformis

The cytoplasmic and chloroplast ribosomes from the marine diatom Cylindrotheca fusiformis were isolated and characterized. The cytoplasmic ribosomes sedimented in sucrose at 84S and dissociated into subunits of 64S and 42S in the absence of Mg²⁺. It contained ribosomal RNAs with molecular weights of 1.31×10⁶ and 0.70×10⁶. The chloroplast ribosomes sedimented at 70S only in the presence of high Mg²⁺ concentrations (25–100 mM). No stable subunits were routinely observed and at very high levels of Mg²⁺ (>100 mM) the 70S species was converted to a form sedimenting at 55S. At 4°C ribosomal RNAs with molecular weights of 1.1×10⁶ and 0.40×10⁶ were detected on polyacrylamide gel electrophoresis. When the RNAs were resolved at room temperature the large molecular weight component disappeared while RNA with molecular weights of 0.65×10⁶ and 0.53×10⁶ were observed. Apparently the large chloroplast RNAs dissociated into two pieces of unequal molecular weight. These properties of the diatom's chloroplast ribosomes are very similar to those of the counter parts in unicellular green algae, which suggests that both types of algae have a common phylogenetic ancestor.     

Role of polyamines in the binding of initiator tRNA to the 70S ribosomes of extreme thermophilic bacterium Calderobacterium hydrogenophilum

Slowly cooled cells of an extreme thermophilic eubacterium Calderobacterium hydrogenophilum possess ribosomes with weakly associated subunits. These ribosomal subunits are capable of association to 70S ribosomes either at higher Mg²⁺ concentrations (30–40 mM) or at 4–10 mM Mg²⁺ and in the presence of polyamines. The contribution of 30S and 50S subunits to the hydrodynamic stability of ribosomes was examined by forming hybrid 30S–50S couples from C. hydrogenophilum and Escherichia coli. At lower Mg²⁺ (4–10 mM) heterogeneous subunits containing 30S E. coli and 50S C. hydrogenophilum and homogeneous subunits of the thermophilic bacterium associated only in the presence of polyamines. Ribosomal subunits associated at 30 mM Mg²⁺ lose thermal stability and activity concerning poly(AUG)-dependent binding of f[³H]Met-tRNA to the P-site on 70S ribosomes or translation of poly(UG). Poly(AUG), deacylated-tRNA or initiator-tRNA have no valuable effect on association of 30S and 50S subunits. Protein synthesis initiation factor IF3 of C. hydrogenophilum prevents association of ribosomal subunits to 70S ribosomes at physiological temperature (70°C). The factor also stimulates dissociation of 70S ribosomes of E. coli at 37°C. The codon-specific binding of f[³H]Met-tRNA to homogeneous 70S ribosomes of C. hydrogenophilum at 70°C is dependent on the presence of initiation factors and concentrations of tri-pentaamines. However, excess of polyamines inhibited the reaction. Our results indicate that tri-pentaamines enhance conformational stability of 70S initiation complex at elevated temperatures.     

Studies of Ribosomal Diffusion Coefficients Using Laser Light-Scattering Spectroscopy

Using an optical beating technique, the diffusion coefficients and relative scattered intensity of Escherichia coli 70S, 50S, and 30S ribosomes are measured as a function of temperature and Mg²⁺ concentration. For solutions at 10 mM Mg²⁺ and between 0°C and about 40°C, the values of D_20,w obtained are 1.7, 1.9, and ≈2.1 × 10^-7 cm²/s, respectively. Preparative procedures drastically affect these values and equivalent hydrodynamic ellipsoids of revolution models give large axial ratios indicating extensive hydration or a deviation from the assumed shape. Calculations also indicate that the subunits expand upon dissociation. Measurements of D_20,w vs. temperature indicate that 70S particles undergo a conformational change prior to dissociation and can be heat dissociated at 30-32°C at low concentrations. Treatment of 70S ribosomes with EDTA causes a biphasic dissociation reaction. Addition of Mg²⁺ after dissociation with EDTA shows that longer waiting times yield fewer 70S particles and that even short waiting times may yield ribosomes differing from the native conformation. Addition of p-chloromercuribenzoic acid (PCMB) is shown to dissociate 70S particles, but to a lesser extent than ethylenediaminetetraacetic acid (EDTA).     

Specificity and kinetics of 23S rRNA modification enzymes RlmH and RluD

Along the ribosome assembly pathway, various ribosomal RNA processing and modification reactions take place. Stem–loop 69 in the large subunit of Escherichia coli ribosomes plays a substantial role in ribosome functioning. It contains three highly conserved pseudouridines synthesized by pseudouridine synthase RluD. One of the pseudouridines is further methylated by RlmH. In this paper we show that RlmH has unique substrate specificity among rRNA modification enzymes. It preferentially methylates pseudouridine and less efficiently uridine. Furthermore, RlmH is the only known modification enzyme that is specific to 70S ribosomes. Kinetic parameters determined for RlmH are the following: The apparent K_M for substrate 70S ribosomes is 0.51 ± 0.06 μM, and for cofactor S-adenosyl-L-methionine 27 ± 3 μM; the k_cat values are 4.95 ± 1.10 min⁻¹ and 6.4 ± 1.3 min⁻¹, respectively. Knowledge of the substrate specificity and the kinetic parameters of RlmH made it possible to determine the kinetic parameters for RluD as well. The K_M value for substrate 50S subunits is 0.98 ± 0.18 μM and the k_cat value is 1.97 ± 0.46 min⁻¹. RluD is the first rRNA pseudouridine synthase to be kinetically characterized. The determined rates of RluD- and RlmH-directed modifications of 23S rRNA are compatible with the rate of 50S assembly in vivo. The fact that RlmH requires 30S subunits demonstrates the dependence of 50S subunit maturation on the simultaneous presence of 30S subunits.     

Binding of spectinomycin by ribosomes fromEscherichia coli

The binding of the aminocyclitol antibiotic spectinomycin to 70S ribosomes and to 30S subunits fromEscherichia coli has been investigated. The association was influenced by the presence of messenger RNA. The K_d for [³H]-4 OH-spectinomycin binding to 70S ribosomes was 2×10^–7 M without mRNA (polyinosinic acid), and 1×10^–6 M with polyinosinic acid. Dissociation of the antibiotic from the ribosomes was significantly affected by the presence of a bound messenger RNA, which reduced the rate of dissociation by a factor of 5.7. The presence of mRNA did not influence the association of spectinomycin with the 30S subunit. The dissociation rate from the small subunit was comparable to the rate of dissociation from the 70S ribosome and was not affected by the presence of mRNA.     

Physicochemical and Light Scattering Studies on Ribosome Particles

The light scattering technique has been used to measure the molecular weight of Escherichia coli ribosomes. The 30S, 50S, and 70S components have been isolated and purified. The refractive index increment dn/dc was found to have the same value, (0.20 ±0.01) cm³/g, for the three species. The molecular weights are (1.0 ±0.1)·10⁶, (1.7 ±0.1)·10⁶, and (2.9 ±0.3)·10⁶ daltons respectively. Some information about the dimensions in solution (radius of gyration) and the interaction constant (second virial coefficient) have been obtained, and their significance is discussed.     

Gel electrophoresis of ribosomal components from seeds of Pisum sativum L

The ribosomes of dry pea seeds were analysed by polyacrylamide gel electrophoresis. Ribosomes, ribosomal subunits, rRNA and ribosomal proteins were separated by variations of this same basic technique. Pea seed ribosomes were shown to have a subunit structure, rRNA complement and ribosomal protein distribution similar to other eukaryotic ribosomes. A total of 52 ribosomal proteins were identified, 24 on the small and 28 on the large RSU. The molecular weights were mostly in the range 10–35 × 10³.     

Synthesis of Double-Stranded RNA in a Virus-Enriched Fraction from Agaricus bisporus

Partially purified virus preparations from sporophores of Agaricus bisporus affected with LaFrance disease had up to a 15-fold-higher RNA-dependent RNA polymerase activity than did comparable preparations from healthy sporophores. Enzyme activity was dependent upon the presence of Mg²⁺ and the four nucleoside triphosphates and was insensitive to actinomycin D, α-amanitin, and rifampin. The ³H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 × 10⁶ and 1.4 × 10⁶; they corresponded in size and hybridized to the major dsRNAs detected in the virus preparation by ethidium bromide staining. Cs₂SO₄ equilibrium centrifugation of the virus preparation resolved a single peak of RNA polymerase activity that banded with a 35-nm spherical virus particle containing dsRNAs with molecular weights of 4.3 × 10⁶ and 1.4 × 10⁶. The data suggest that the RNA-dependent RNA polymerase associated with the 35-nm spherical virus is a replicase which catalyzes the synthesis of the genomic dsRNAs.     

Characterization of ribosomes of a strain ofStreptomyces aureofaciens producing chlortetracycline

These studies were designated to investigate the effect of chlortetracycline on sedimentation properties of polysomes and ribosomes present in the chlortetracycline producing strain ofStreptomyces aureofaciens. In presence of chlortetracycline polysomes and ribosomes are more stable than the bacterial ones. At lower chlortetracycline concentrations (1–5 μg/ml) dissociation of polysomes into 70 S monomers was not observed. Ribosomes in higher concentration of chlortetracycline (400 μg/ml) form aggregates. A decrease of Mg²⁺ to 0.1mm caused dissociation of ribosomes to two subunits and in this state none of indicated concentrations of chlortetracycline caused aggregation. The exact sedimentation values of ribosomes and ribosomal subunits were calculated from extrapolation to infinite dilution. S_20,w for monomer form was 68.8, and for ribosomal subunits 49.8 and 31.2 respectively. Ribosomal RNA sedimentates as two Schlieren peaks of 16 S and 22 S. It was found that 30 S subunits contain 15 structural proteins, while 21 proteins were resolved from 50 S subunits.     

Studies on the interaction between ribosomes and 14 CH 3 -F 3 initation factor

Summary Initiation factor F₃ has been purified fromEscherichia coli and labelledin vitro by reductive alkylation. The¹⁴CH₃–F₃ so obtained had a specific activity of about 1 000 cpm/g and was shown to have retained its biological activity. Labelled F₃ binds to 30S ribosomal subunits ofEscherichia coli andBacillus stearothermophilus, but does not bind to either 70S ribosomes or 50S ribosomal subunits. The stoichiometry of the binding indicates that one molecule of¹⁴CH₃–F₃ is bound to each 30S ribosomal subunit. Several antibiotics, known to interact with 30S subunits, inhibit the binding. Functional studies indicate that F₃ is released from 30S ribosomes as a result of the formation of the 70S initiation complex.     

ISOLATION OF CYTOPLASMIC AND CHLOROPLAST RIBOSOMES AND THEIR DISSOCIATION INTO ACTIVE SUBUNITS FROM CHLAMYDOMONAS REINHARDTII

A mixture of cytoplasmic (80S) and chloroplast (70S) ribosomes from Chlamydomonas reinhardtii was freed of contaminating membranes by sedimentation of the postmitochondrial supernatant through a layer of 1.87 M sucrose. The purified ribosomes were separated into 80S and 70S fractions by centrifugation at a relatively low speed on a 10–40% sucrose gradient containing 25 mM KCl and 5 mM MgCl₂. Both the 80S and 70S ribosomes were dissociated into compact subunits by centrifugations in 5–20% high-salt sucrose gradients. The dissociations of both ribosomal species under these conditions were not affected by the addition of puromycin, indicating that the ribosomes as isolated were devoid of nascent chains. Subunits derived from the 80S ribosomes had apparent sedimentation coefficients of 57S and 37S whereas those from the 70S ribosomes had apparent sedimentation coefficients of 50S and 33S. In the presence of polyuridylic acid and cofactors, the 80S and 70S ribosomes incorporated [¹⁴C]phenylalanine into material insoluble in hot TCA. The requirements for incorporation were found to be similar to those described for eukaryotic and prokaryotic ribosomes. Experiments with antibiotics showed that the activity of the 80S ribosomes was sensitive to cycloheximide, whereas that of the 70S ribosomes was inhibited by streptomycin. The isolated subunits, when mixed together in an incorporation medium, were also active in the polymerization of phenylalanine in vitro.     

Changes in the conformation of 5S rRNA cause alterations in principal functions of the ribosomal nanomachine

5S rRNA is an integral component of the large ribosomal subunit in virtually all living organisms. Polyamine binding to 5S rRNA was investigated by cross-linking of N¹-azidobenzamidino (ABA)-spermine to naked 5S rRNA or 50S ribosomal subunits and whole ribosomes from Escherichia coli cells. ABA-spermine cross-linking sites were kinetically measured and their positions in 5S rRNA were localized by primer extension analysis. Helices III and V, and loops A, C, D and E in naked 5S rRNA were found to be preferred polyamine binding sites. When 50S ribosomal subunits or poly(U)-programmed 70S ribosomes bearing tRNA^Phe at the E-site and AcPhe-tRNA at the P-site were targeted, the susceptibility of 5S rRNA to ABA-spermine was greatly reduced. Regardless of 5S rRNA assembly status, binding of spermine induced significant changes in the 5S rRNA conformation; loop A adopted an apparent ‘loosening’ of its structure, while loops C, D, E and helices III and V achieved a more compact folding. Poly(U)-programmed 70S ribosomes possessing 5S rRNA cross-linked with spermine were more efficient than control ribosomes in tRNA binding, peptidyl transferase activity and translocation. Our results support the notion that 5S rRNA serves as a signal transducer between regions of 23S rRNA responsible for principal ribosomal functions.     

Specific binding of ribosome recycling factor (RRF) with the Escherichia coli ribosomes by BIACORE

The direct assays on Biacore with immobilised RRF and purified L11 from E. coli in the flow trough have shown unspecific binding between the both proteins. The interaction of RRF with GTPase domain of E. coli ribosomes, a functionally active complex of L11 with 23S r RNA and L10.(L7/L12)₄ was studied by Biacore. In the experiments of binding of RRF with 30S, 50S and 70S ribosomes from E. coli were used the antibiotics thiostrepton, tetracycline and neomycin and factors, influencing the 70S dissociation Mg²⁺, NH₄Cl, EDTA. The binding is strongly dependent from the concentrations of RRF, Mg²⁺, NH₄Cl, EDTA and is inhibited by thiostrepton. The effect is most specific for 50S subunits and indicates that the GTPase centre can be considered as a possible site of interaction of RRF with the ribosome. We can consider an electrostatic character of the interactions with most probable candidate 16S and 23S r RNA at the interface of 30S and 50S ribosomal subunits.     

Characterization of different conformational forms of 30 S ribosomal subunits in isolated and associated states: possible correlations between structure and function.

The reaction pattern with N-[¹⁴C]ethylmaleimide served to follow conformational changes of 30 S ribosomal subunits that are induced by association with 50 S subunits and by the binding of aminoacyl-tRNA to 70 S ribosomes either enzymatically or non-enzymatically.The usefulness of the reaction with N-ethylmaleimide in discerning different conformational forms of the ribosome was previously demonstrated (Ginzburg et al., 1973) in an analysis of inactive and active 30 S subunits (as obtained at low Mg²⁺ and after heat reactivation, respectively). The reaction pattern of the 30 S moiety of 70 S ribosomes differs from the pattern of isolated active subunits (the only form capable of forming 70 S ribosomes) in both the nature of the labeled proteins and in being Mg²⁺-dependent. The reaction at 10 mm-Mg²⁺ reveals the following differences between isolated and reassociated 30 S subunits: (1) proteins S1, S18 and S21 that are not labeled in isolated active subunits, but are labeled in the inactive subunits, are highly reactive in 70 S ribosomes; (2) proteins S2, S4, S12 and S17 that uniquely react with N-ethylmaleimide in active subunits are all rendered inaccessible to modification after association; and (3) proteins S9, S13 and S19, that react in both active and inactive 30 S subunits, are labeled to a lesser extent in the 70 S ribosomes than in isolated subunits. This pattern is altered in two respects when the reaction with the maleimide is carried out at 20 mm-Mg²⁺; protein S18 is not modified while S17 becomes labeled.The differences in reaction pattern are considered as manifesting the existence of different conformational forms of the 30 S subunit in the dissociated and associated states as well as of different forms of 70 S ribosomes. The 30 S moiety of 70 S ribosomes at 10 mm-Mg²⁺ resembles the inactive subunit, while some of the features of the active subunit are preserved in the 70 S ribosome at 20 mmMg²⁺. The structural changes appear to be expressed in the functioning of the ribosome: non-enzymatic binding of aminoacyl-tRNA to active 30 S subunits is suppressed by 50 S subunits at 10 mm but not at 20 mm-Mg²⁺ (Kaufmann &; Zamir, 1972). The fact that elongation factor Tu-mediated binding is not suppressed by 50 S subunits raises the possibility that the function of the elongation factor might involve the facilitation of a conformational change of the ribosome. The analysis of different ribosomal binding complexes with N-ethylmaleimide showed that the binding of poly(U) alone results in a decrease in the labeling of S1 and S18. Binding of aminoacyl-tRNA, on the other hand, is closely correlated with the exposure of S17 for reaction with the maleimide. A model is outlined that accounts for this correlation as well as for the proposed role of elongation factor Tu.     

The identification of spermine binding sites in 16S rRNA allows interpretation of the spermine effect on ribosomal 30S subunit functions

A photoreactive analogue of spermine, N¹-azidobenzamidino (ABA)-spermine, was covalently attached after irradiation to Escherichia coli 30S ribosomal subunits or naked 16S rRNA. By means of RNase H digestion and primer extension, the cross-linking sites of ABA-spermine in naked 16S rRNA were characterised and compared with those identified in 30S subunits. The 5′ domain, the internal and terminal loops of helix H24, as well as the upper part of helix H44 in naked 16S rRNA, were found to be preferable binding sites for polyamines. Association of 16S rRNA with ribosomal proteins facilitated its interaction with photoprobe, except for 530 stem–loop nt, whose modification by ABA-spermine was abolished. Association of 30S with 50S subunits, poly(U) and AcPhe-tRNA (complex C) further altered the susceptibility of ABA-spermine cross-linking to 16S rRNA. Complex C, modified in its 30S subunit by ABA-spermine, reacted with puromycin similarly to non-photolabelled complex. On the contrary, poly(U)-programmed 70S ribosomes reconstituted from photolabelled 30S subunits and untreated 50S subunits bound AcPhe-tRNA more efficiently than untreated ribosomes, but were less able to recognise and reject near cognate aminoacyl-tRNA. The above can be interpreted in terms of conformational changes in 16S rRNA, induced by the incorporation of ABA-spermine.     

CYTOCHEMICAL STUDY ON THE PANCREAS OF THE GUINEA PIG : VII. Effects of Spermine on Ribosomes

Pancreatic ribosomes (guinea pig) aggregate and lose upon treatment with polyamines, particularly spermine, their bound secretory enzymes. Spermine, at 0.5 mM, for example, causes the release of about 85 per cent of the chymotrypsinogen and RNase, and from 85 to 100 per cent of the ribosomal amylase. At the same time, the particles lose about 10 per cent of their RNA, 7 to 24 per cent of their total protein, and from 75 to 100 per cent of their Mg⁺⁺. Observations with the electron microscope confirm the heavy agglutinating of the ribosomes but otherwise show little change in the structure of the particles. Using radioactive spermine it was found that, concomitant with the loss of bound enzymes and Mg⁺⁺ from the ribosomes, spermine became bound to the particle. The extent of binding ranged from 0.29 to 1.49 µmoles per 10µmoles RNA-P. The bound radioactive spermine can be removed by subsequent treatment of the ribosomes with GTP, ATP, or P-P, which treatment also removes most of the RNA of the particles, leaving behind ribosomes with a much lower RNA/protein ratio. From this evidence it was inferred that spermine, in releasing the Mg⁺⁺ of the particle, becomes salt-linked to the free phosphate hydroxyl groups of the RNA. Freshly isolated pancreatic and hepatic ribosomes contain very little spermine, about 0.1 to 0.2 µmoles polyamine/10 µmoles RNA-P. The results are discussed in terms of the linkages between the structural protein, the bound secretory enzymes, and the RNA of the ribosomes.