Binding of [14C]tuberactinomycin O, an antibiotic closely related to viomycin, to the bacterial ribosome.

The binding of [¹⁴C]tuberactinomycin O, an antibiotic closely related to viomycin, to $\text{E. coli}$ ribosomes has been examined by equilibrium dialysis method. The antibiotic has been observed to bind to the 70S ribosome, which possesses two binding sites: one on the 30S ribosomal subunit and another on the 50S subunit. The affinity for the large subunit is greater than that for the small subunit. The binding to both ribosomal subunits is reversed by viomycin, indicating that tuberactinomycin O and viomycin have the same binding sites on the ribosome. The results seem to be in accordance with the previous finding that viomycin exhibits dual actions on ribosomal function: the inhibition of fMet-tRNA_F (initiation) and inhibition of translocation of peptidyl-tRNA.     

Interactions between viomycin resistance and streptomycin resistance on ribosomes of Mycobacterium smegmatis.

We have investigated the mechanism of the expression of resistance to high levels of viomycin and coresistance to streptomycin in a mutant strain of Mycobacterium smegmatis ATCC 14468 (AC-13) which was obtained by serial transfers of parental cells to media containing increasing concentrations of viomycin. It was shown previously that resistance to viomycin by strain AC-13 was due to an alteration in the 50 S ribosomal subunit (20). However, genetic analysis has shown that mutation in 50 S subunits alone gave only low level resistance to viomycin. When a streptomycin resistant mutation (caused by an alteration in the 30 S subunit) was introduced into the low level viomycin resistant recombinant strains, most of them were highly resistant to viomycin. Some recombinants were resistant to intermediate levels of viomycin, and the remainder were not affected by the introduction of the str^r allele. Studies with in vitro cell-free systems have shown that streptomycin resistant 30 S ribosomal subunits obtained from a high level viomycin resistant recombinant were able to modify the levels of resistance to viomycin expressed by the 50 S ribosomal subunit. These findings provide additional evidence concerning the functional relationship between 30 S and 50 S ribosomal components in ribosomes.     

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.     

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 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.     

Action of methanol on the assocation of ribosomal subunits and its effect on the GTPase activity of elongation factor G

Methanol causes association of 30S and 50S ribosomal subunits from $\text{E. coli}$ at MgCl₂ concentrations in which they are normally completely dissociated. The 70S ribosome formed under these conditions shows a lower sedimentation velocity and is functionally active in the EF-G GTPase. Association of ribosomal subunits in the presence as well as absence of methanol is affected by washing the ribosomes with 0.5 M NH₄Cl. Methanol reduces the Mg²⁺ concentration required for subunit association as well as for EF-G GTPase activity. The basic requirement for EF-G GTPase activity both with and without alcohol is shown to be the association of 30S and 50S subunits.     

The effect of both homologous and heterologous DNA on integration efficiencies in pneumococcal transformation

Summary A mutant of the yeast Saccharomyces cerevisiae has been isolated that is resistant to narciclasine, an inhibitor of peptide bond formation on 80S ribsomes. The mutant shows cross-resistance to a number of inhibitors of peptidyl transferase including anthelmycin, a 4-aminohexosyl cytosine antibiotic, which does not compete with narciclasine for its ribosomal binding site. The mutation is within the gene tcm1 or a closely linked gene on chromosome XV; it is expressed in the 60S ribosomal subunit. The parameters of the binding of (³H)narciclasine to ribosomes and ribosomal subunits from both wild-type and mutant strains have been calculated by ultracentrifugation. One molecule of narciclasine is bound per ribosome or per 60S ribosomal subunit, the values of the dissociation constants being 0.054 and 0.13 m respectively, for 80S and 60S particles from the wild-type cells. Ribosomes of the mutant strain have a lower affinity for narciclasine and trichodermin than ribosomes from wild-type cells. The mutation is semidominant in heterozygous diploid cells.     

Antagonistic action between spermidine and putrescine on association and dissociation of purified, run-off ribosomes from Escherichia coli.

The effects of polyamines on the equilibrium between prokaryotic ribosomal subunits and 70 S ribosomes have been studied as a function of concentration of Mg2+ from 2.5 to 7.5 mM. Run-off ribosomes were obtained from Escherichia coli and were washed with buffered 1 M NH4C1. Spermidine at 1 mm favors association of subunits at all concentrations of Mg2+. Putrescine, at concentrations above 8 mM, favors net dissociation at concentrations of Mg2+ below 4.5 mM. Streptomycin behaves like spermidine, while putrescine behaves like initiation factor 1 and initiation factor 3. The effect of putrescine on dissociation is time-dependent and appears to have a half-life of about 3.5 min at 30 degrees. When added after the effects of spermidine or streptomycin on association have occurred, putrescine still causes dissociation. The data suggests that putrescine may reduce net formation of vacant 70 S ribosomes. Another possibility is that putrescine and spermidine may act antagonistically to maintain a labile equilibrium between ribosomal subunits and vacant 70 S ribosomes. It may be significant that the putrescine effect is observed at the concentration of Mg2+ found to be optimum for initiation.     

Dual actions of viomycin on the ribosomal functions.

Viomycin was observed to inhibit poly[U]- or f2 RNA-directed protein synthesis in an $\text{E. coli}$ cell-free system. The former was more profoundly affected than the latter. Both initiation complex formation on the 30S ribosomal subunit and on 70S ribosomes were prevented by the antibiotic. In the peptide chain elongation process, viomycin did not significantly affect aminoacyl-tRNA binding to ribosomes and the peptidyl transferase reaction, but markedly inhibit translocation of peptidyl-tRNA from the acceptor site to the donor site. The mechanism of action of the drug appeared to be unique.     

Proteins occurring at, or near, the subunit interface of E. coli ribosomes

Summary The identification of ribosomal proteins that occur at, or near, the subunit interface of the 30S and 50S subunits in the E. coli 70S ribosome was attempted by studying the effect of antibodies on the Mg⁺⁺ dependent dissociation-association equilibrium of 70S ribosomes. Dissociated ribosomes were mixed with monovalent fragments of IgG antibodies (Fab's) specific for each ribosomal protein and then reassociated into intact 70S particles. Various degrees of inhibition of this reassociation were observed for proteins S9, S11, S12, S14, S20, L1, L6, L14, L15, L19, L20, L23, L26 and L27. A small amount of aggregation of 50S subunits was caused by IgG's specific for the proteins S9, S11, S12, S14 and S20 and purified 50S subunits. It was inferred that the presence of small amounts of these proteins on 50S subunits was compatible with their presence at the subunit interface. Finally, the capacity of proteins S11 and S12 to bind to 23S RNA was demonstrated.Paper No. 84 on Ribosomal Proteins. Preceding paper is by Rahmsdorf et al., Molec. gen. Genet. 127, 259–271 (1973).     

Characterization of spinach plastid ribosomal proteins by two dimensional gel electrophoresis

Summary Ribosomes are isolated from spinach plastids using conventional sucrose gradients. Their subunits are prepared by dissociation using low Mg²⁺ concentration.It is shown that plastid ribosomes are able to bind f-met-tRNA in the presence of the initiation factors from E. coli.The characterization of ribosomal proteins is carried out using the four two-dimensional gel electrophoretic systems of Madjar et al. (1979). The 30 S and 50 S subunits contain 24 and 34 ribosomal poteins, respectively. These proteins are found in the 70S monosomes which also contain most often nine additional faintly stained proteins.     

Loosening and unfolding of E. coli 50 S ribosomal subunits: Dependence on magnesium content and temperature

Reversible change of 50 S ribosomal subunits to 40 S particles takes place in cold buffered 0.5 M NH₄Cl solutions either containing Mg⁺⁺ (up to 0.1 M), or free from Mg⁺⁺ and even supplemented with EDTA (1 mM). The 40 S particles were stable only within a definite temperature range. Heating of the samples caused completely irreversible unflding of the 40 S particles. This melting appeared to be co-operative and took place within a very narrow range of temperature, which for samples containing Mg⁺⁺ was a linear function of the log of Mg⁺⁺ concentration.The results suggest that two types of bonds maintained the compact structure of the ribosomal subunits: ionic bonds involving Mg⁺⁺ and heat-labile weak interactions between ribosomal components.     

Thermal Analysis of Bacterial Ribosomes

Ribosomal particles of E. coli were examined by using a heat leakage scanning calorimeter. Remarkable changes were observed in thermograms of 70S ribosomes and their subunits when the Mg²⁺ concentration was raised from 1 mm to 10 mm. It was suggested that ribosomal subunits exist in more than one conformation, and changes in their conformation might be the primary cause of the association-dissociation process of ribosomes. Comparisons of thermograms of RNase- and chymotrypsin-treated, as well as non-treated SOS and 30S subunits suggest that conformational changes in each subunit may be ascribed to changes in rRNA.     

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.     

Ribosomal protein as substrate for a GTP-dependent protein kinase from yeast

A protein kinase specific for casein and acidic ribosomal proteins was isolated and partly characterized.It was found that the enzyme utilizes GTP and ATP as phosphoryl donors. Its affinity for ATP was considerably higher than for GTP with the km values of 7.6 × 10^-6M and 5.5 × 10^-5M, respectively.Two-dimensional acrylamide gel electrophoresis revealed the phosphorylation of the same ribosomal proteins with either of the [-³²P] nucleotides used. It was also shown that one acidic protein (S1 or S2) of 40 S and two acidic proteins (L2 and L3) of 60 S ribosomal subunits were predominantly phosphorylated in vitro. The phosphorylated proteins: L2 and L3 seem to correspond to the proteins of L7 and L12 of E. coli ribosomes. The isolated kinase phosphorylated several basic ribosomal proteins though to a lower extent than the acidic ones.     

Silver Plated Ribosomes

WE recently reported that dissociation of free 70S ribosomes from E. coli to 30S and 50S subunits could occur during sedimentation in buffer containing K⁺ as the predominant monovalent cation¹. Spermidine at a physiological concentration (1 mM) specifically prevented the dissociation and we suggested that the dissociation that occurred in its absence was due to loss of the polyamine from the 70S ribosome. We have subsequently found that this hypothesis is wrong and that the dissociation that took place in the original experiments was due not primarily to loss of spermidine, but to a reaction with silver present in very small amounts in the AR KCl used in the buffer.     

Ribosome activity and modification of 16S RNA are influenced by deletion of ribosomal protein S20

A spontaneous mutant of Escherichia coli K-12 was isolated that shows an increased misreading ability of all three nonsense codons together with an inability to grow at 42° C. It is demonstrated that the mutation is a deletion of the gene rpsT, coding for ribosomal protein S20. The loss of this protein not only influences the decoding properties of the ribosome; the modification pattern of 16S ribosomal RNA is also changed. This leads to a deficiency in the ability of the mutant to associate its 30S subunits with 50S subunits to form 70S ribosomes. It is suggested that two modified bases, m⁵C and m⁶₂A, are directly or indirectly essential for association of subunits to functional ribosomes in the rpsT mutant strain. Two other modifications were also studied; m²G which is not affected at all and m³U which is undermodified in both active and inactive subunits and, therefore, not involved in subunit association.     

Analysis of Ribosomes from Viomycin-Sensitive and -Resistant Strains of Mycobacterium smegmatis

Viomycin-resistant strains were isolated from Mycobacterium smegmatis. Ribosomes were isolated and tested for drug resistance in subcellular systems containing poly(U) as messenger ribonucleic acid. Resistance to viomycin in these strains was due to altered ribosomes. Further analysis showed that viomycin resistance of two mutants with low level resistance (20 mug/ml) was due to altered 30S ribosomal subunits. Another mutant that was highly resistant to viomycin (1 mg/ml), however, had altered 50S ribosomal subunits.     

Letter: Conformational changes in ribosomal subunits following dissociation of the Escherichia coli 70 S ribosome

The reactivity of various Escherichia coli ribosomal proteins with N-ethylmaleimide has been used as a probe for ribosomal topography changes during the subunit-70 S transition. With the 70 S ribosome there are several proteins from both subunits which do not react with N-ethylmaleimide, but which do so after dissociation of the 70 S particle to free 30 S and 50 S subunits. The kinetics of their exposure is slow relative to that of the 70 S dissociation reaction suggesting conformational changes in both subunits subsequent to 70 S particle dissociation.