Isolation of ribosomal subparticles from human placenta containing intact rRNA and determination of the functional activity of the 80S ribosome

The method for isolation of human placenta ribosomal subunits containing intact rRNA has been determined. The method uses fresh unfrozen placenta. Activity of 80S ribosomes obtained via reassociation of 40S and 60S subunits in non-enzymatic poly(U)-mediated Phe-tRNAPhe binding, was near 75% (maximal [14C]Phe-tRNA(Phe) binding was 1.5 mol Phe-tRNA(Phe) per mol of 80S ribosomes). Activity of 80S ribosomes with damaged rRNA isolated from frozen placenta was 2 times lower (the maximum level of poly(U)-dependent Phe-tRNA(Phe) binding was 0.7 mol per mol of ribosomes). The activity 80S ribosomes in poly(U)-mediated synthesis of polyphenylalanine was determined by using fractionated ("ribosomeless") protein synthesising system from rabbit reticulocytes. In this system up to the 50 mol of Phe residues per mol of 80S ribosomes are incorporated in acid insoluble fraction in 1 hour, at 37 degrees C. The obtained level of [14C]phenylalanine incorporation is three times as much as the amount of Phe residues observed for the ribosomal subunits, isolated from frozen placenta.     

Isolation of ribosomal subunits containing intact rRNA from human placenta: estimation of functional activity of 80S ribosomes.

We have elaborated a method for the isolation of ribosomal subunits from fresh unfrozen human placenta containing intact rRNA and a complete set of ribosomal proteins. Activity of 80S ribosomes obtained by reassociation of 40S and 60S subunits in nonenzymatic poly(U)-dependent binding of Phe-tRNA(Phe) was equal to 80% (above 1.5 mol [14C]Phe-tRNA(Phe) is coupled to 1 mol of ribosomes). The activity of 80S ribosomes in poly(U)-directed synthesis of polyphenylalanine was tested in a polysome-free protein-synthesizing system from rabbit reticulocytes. About 100 mol of phenylalanine residue was polymerized by a mole of ribosomes at a rate of 0.83 residues per minute in this system (2 h, 37 degrees C).     

Biochemical changes in hereditary progressive muscular dystrophies. Defect of protein synthesis in fibroblasts, muscle tissues and blood cells

80S ribosomes and ribosomal subunits were isolated from fibroblasts, muscle tissues and blood cells of patients with different muscular dystrophies (MD) as well as of controls and were used for in vitro measurement of ribosomal protein synthesis (RPS) in a poly(U)-directed polyphenylalanine synthesis system. The activity of ribosomes from the patients showed a disease-dependent decrease compared to normal controls. Examination of hybrid 80S ribosomes consisting of 40S and 60S subunits of patients and the corresponding control cells revealed that the loss of RPS activity was related to one or both of the ribosomal subunits depending on the type of MD.     

Reassociation of cytoplasmic ribosomal subunits of barley and its virescens mutant to form active hybrids

Polyphenylalamine synthesis by cytoplasmic ribosomes of Gateway barley (Hordeum vulgare) and its virescens single gene nuclear mutant was compared. The cytoplasmic 80S ribosomes were isolated from unimbibed embryo material and the ribosomes were dissociated into their component 60S and 40S subunits by centrifugation through sucrose gradients containing high KCl-to-MgCl₂ buffer. These separated subunits could be reassociated by resuspension in buffer having about equimolar concentrations of MgCl₂ and KCl. Both homologous and heterologous combinations of the subunits reassociated to give monomeric 80S ribosomes, and the derived monomers as well as various combinations of the individual subunits showed equivalent activity in an in vitro system for poly (U)-directed polyphenylalanine synthesis.     

Translation of Synthetic and Endogenous Messenger Ribonucleic Acid In Vitro by Ribosomes and Polyribosomes from Clostridium pasteurianum

Ribosomes and polyribosomes from Clostridium pasteurianum were isolated and their activities were compared with those of ribosomes from Escherichia coli in protein synthesis in vitro. C. pasteurianum ribosomes exhibited a high level of activity due to endogenous messenger ribonucleic acid (RNA). For translation of polyuridylic acid [poly(U)], C. pasteurianum ribosomes required a higher concentration of Mg(2+) and a much higher level of poly(U) than did E. coli ribosomes. Phage f2 RNA added to the system with C. pasteurianum ribosomes gave no significant stimulation of protein synthesis in a homologous system or with E. coli initiation factors. The 30S and 50S subunits prepared from C. pasteurianum ribosomes reassociated less readily than subunits from E. coli. The ability of the C. pasteurianum subunits to reassociated was found to be dependent upon the presence of a reducing agent during preparation and during analysis of the reassociation products. In heterologous combinations, E. coli 30S subunits associated readily with C. pasteurianum 50S subunits to form 70S particles, but C. pasteurianum 30S subunits and E. coli 50S subunits did not associate. In poly(U) translation, E. coli 30S subunits were active in combination with 50S subunits from either E. coli or C. pasteurianum, but C. pasteurianum 30S subunits were not active in combination with either type of 50S subunits. Polyribosomes prepared from C. pasteurianum were very active in protein synthesis, and well-defined ribosomal aggregates as large as heptamers could be seen on sucrose gradients. An attempt was made to demonstrate synthesis in vitro of ferredoxin.     

The effect of mutations in ribosomal proteins S4, S12 and L7/L12 on EF-Tu-dependent expenditure of GTP in the process of codon-specific elongation and misreading of poly(U)

The effect of mutations in ribosomal proteins S4 (rpsD12), S12 (rpsL282) and L7/L12 (rplL265) of Escherichia coli K12 on the EF-Tu-dependent expenditure of GTP during codon-specific elongation (poly(Phe) synthesis on poly(U] and misreading (poly(Leu) synthesis on poly(U], was studied. Under the conditions used the mutations in proteins S4 and L7/L12 did not practically affect the EF-Tu-dependent expenditure of GTR during the poly(Phe) synthesis on poly(U): the GTP/Phe ratio was about 1, as in the case of the wild strain. Under the same conditions, the ribosomes with a mutant S12 protein tended to discard some amount of Phe-tRNA, as a result of which the GTP/Phe ratio increased to about 3. The marked inhibition of misreading by ribosomes with a mutant S12 protein was accompanied by a significant increase of GTP expenditure at the stage of EF-Tu-dependent non-cognate aminoacyl-tRNA binding. In mutant S 12 proteins the GTP/Leu ratio was about 30-40, whereas in the wild type it was about 12. In contrast, stimulation of misreading by ribosomes with mutant S4 and L7/L12 proteins was accompanied by a decrease of the EF-Tu-dependent expenditure of GTP by 2-3 GTP molecules per one Leu residue included into the peptide.     

Preparation of Escherichia coli 70S ribosomes labeled with 35S

[35S]--70S ribosomes (150 Ci/mmol) were isolated from E. coli MRE-600 cells grown on glucose-mineral media in the presence of [35S] ammonium sulfate. The labeled 30S and 50S subunits were obtained from [35S] ribosomes by centrifugation in a sucrose density gradient of 10--30% under dissociating conditions (0.5 mM Mg2+). The activity of [35S]--70S ribosomes obtained by reassociation of the labeled subunits during poly(U)-dependent diphenylalanine synthesis was not less than 70%. The activity of [35S]--70S ribosomes during poly(U)-directed polyphenylalanine synthesis was nearly the same as that of the standard preparation of unlabeled ribosomes. The 23S, 16S and 5S RNAs isolated from labeled ribosomes as total rRNA contained no detectable amounts of their fragments as revealed by polyacrylamide gel electrophoresis. The [35S] ribosomal proteins isolated from labeled ribosomes were analyzed by two-dimensional gel electrophoresis. The [35S] label was found in all proteins, with the exception of L20, L24 and L33 which did not contain methionine or cysteine residues.     

The role of SH-groups of human ribosomal subunits in polypeptide synthesis

The SH-group(s) present in the 60S subunit were shown to play a significant role in the poly U-dependent binding of Phe-tRNA^∗ to 80S ribosomes. 60S subunits preincubated with NEM and assayed with untreated 40S subunits gave only 17% of the normal activity in the non-enzymatic binding of Phe-tRNA and 35% of the normal activity in poly Phe synthesis. NEM-treated 40S plus untreated 60S subunits, however, displayed 77% activity in non-enzymatic binding and 95% activity in poly Phe synthesis.     

Partial release of AcPhe-Phe-tRNA from ribosomes during poly(U)-dependent poly(Phe) synthesis and the effects of chloramphenicol

Poly(U)-programmed 70S ribosomes can be shown to be 80% to 100% active in binding the peptidyl-tRNA analogue AcPhe-tRNA to their A or P sites, respectively. Despite this fact, only a fraction of such ribosomes primed with AcPhe-tRNA participate in poly(U)-directed poly(Phe) synthesis (up to 65%) at 14 mM Mg2+ and 160 mM NH4+. Here it is demonstrated that the apparently 'inactive' ribosomes (greater than or equal to 35%) are able to participate in peptide-bond formation, but lose their nascent peptidyl-tRNA at the stage of Ac(Phe)n-tRNA, with n greater than or equal to 2. The relative loss of early peptidyl-tRNAs is largely independent of the degree of initial saturation with AcPhe-tRNA and is observed in a poly(A) system as well. This observation resolves a current controversy concerning the active fraction of ribosomes. The loss of Ac(Phe)n-tRNA is reduced but still significant if more physiological conditions for Ac(Phe)n synthesis are applied (3 mM Mg2+, 150 mM NH4+, 2 mM spermidine, 0.05 mM spermine). Chloramphenicol (0.1 mM) blocks the puromycin reaction with AcPhe-tRNA as expected but, surprisingly, does not affect the puromycin reaction with Ac(Phe)2-tRNA nor peptide bond formation between AcPhe-tRNA and Phe-tRNA. The drug facilitates the release of Ac(Phe)2-4-tRNA from ribosomes at 14 mM Mg2+ while it hardly affects the overall synthesis of poly(Phe) or poly(Lys).     

Differential features of ribosomes and of poly(U)-programmed cell-free systems derived from sulphur-dependent archaebacterial species

The properties of poly(U)-directed cell-free systems developed from the sulphur-dependent, thermophilic archaebacteria Desulfurococcus mobilis, Thermoproteus tenax, Sulfolobus solfataricus, Thermococcus celer and Thermoplasma acidophilum have been compared. All systems are truly thermophilic in requiring incubation at temperatures close to the physiological optimum for cell growth. Under optimized conditions the error frequency in tRNA selection is less than 0.4% at 80 degrees C, and synthetic efficiencies (Phe residues polymerized per ribosome in 40 min) span from 4 for Tp. tenax, to 10 for Tc. celer, to 20-25 for D. mobilis and T. acidophilum and to 40 for S. solfataricus. According to requirements for polypeptide synthesis and to degree of stability of the ribosomal subunits' association, sulphur-dependent thermophiles cluster into two groups. Group I organisms (D. mobilis, Tp. tenax, S. solfataricus) harbour 70-S monomers composed of weakly associated subunits, whose poly(Phe)-synthesizing capacity is totally dependent on added spermine while being drastically inhibited by monovalent cations. Group II organisms (Tc. celer and T. acidophilum) contain 70-S particles composed of tightly bonded subunits, whose synthetic capacity is independent of spermine while being totally dependent on monovalent cations. Spermine promotes poly(Phe) synthesis on ribosomes of group I organisms by converting the peptidyltransferase center into an active conformation, while monovalent cations are inhibitory by preventing the interaction between the free ribosomal subunits. The closeness between Tc. celer and T. acidophilum ribosomes provides new insight on the phylogenetic placement of Thermococcaceae.     

40 S subunits from rat liver ribosomes contain two codon-dependent sites for transfer RNA

40 S subunits from rat liver ribosomes are able to bind, after heat activation, two molecules of either Phe-tRNAPhe, Ac-Phe-tRNAPhe or deacylated tRNAPhe. Addition of 60 S subunits to the quaternary complex 40 S.poly(U).(Phe-tRNAPhe)2 results in quantitative formation of (Phe)2-tRNAPhe. This indicates that the two binding sites for tRNA on 40 S subunits should be considered as the constituent of P and A sites of 80 S ribosomes.     

The effect of Cephalotaxus group alkaloids on elongation of the polypeptide chain in human ribosomes

Effects of Cephalotaxus alkaloids (homoharringtonine and cephalotaxine) on the translation of endogenous mRNA in a cell-free system of rabbit reticulocyte lysate and on poly(U)-directed poly(Phe) synthesis on human placenta ribosomes was studied. The effect of the alkaloids on the activity of human placenta ribosomes in a template-dependent aminoacyl-tRNA binding, N-acetyl-phenylalanyl-puromycin and diphenylalanine formation was also studied. Homoharringtonine was shown to have little effect of codon-dependent Phe-tRNA(Phe) binding but the alkaloid strongly inhibited (Phe)2 formation as well as N-Ac-Phe-puromycin synthesis from the complex N-Ac-Phe-tRNA(Phe).poly(U).80S ribosomes. It was concluded that the site of homoharringtonine binding overlaps or coincides with the acceptor site of the ribosomal peptidyltransferase center. The association constant of homoharringtonine to the ribosomes was estimated to be (4.8 +/- 1.0) x 10(7) M-1. Cephalotaxine had no effect on the elongation steps.     

Inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin.

The mechanism of inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin was studied. No significant disaggregation of polysomes into monosomes was detected in the toxin-treated cells. The activity of the polysomes isolated from the cells treated with the toxin in protein synthesis was remarkably lower than that of the untreated cells, while the activity of the supernatant enzyme fraction was retained. The ribosomes derived from the polysomes of the toxin-treated cells were inactive in poly(U)-dependent polyphenylalanine synthesis. The activity of ribosomes reconstituted by hybridizing subunits derived from the ribosomes of normal and toxin-treated cells were measured in poly(U)-dependent polyphenylalanine synthesis, and the 60 S subunit was revealed to be inactive. These results indicate that the target of action of the toxin towards intact cells is the 60 S ribosomal subunit.     

Mechanism of codon-anticodon interaction in ribosomes. Direct functional evidence that isolated 30S subunits contain two codon-specific binding sites for transfer RNA.

30S subunits were isolated capable to bind simultaneously two molecules of Phe-tRNAPhe (or N-Acetyl-Phe-tRNAPhe), both poly(U) dependent. The site with higher affinity to tRNA was identified as P site. tRNA binding to this site was not inhibited by low concentrations of tetracycline (2 x 10(-5)M) and, on the other hand, N-Acetyl-Phe-tRNAPhe, initially prebound to the 30S.poly(U) complex in the presence of tetracycline, reacted with puromycin quantitatively after addition of 50S subunits. The site with lower affinity to tRNA revealed features of the A site: tetracycline fully inhibited the binding of both Phe-tRNAPhe and N-Acetyl-Phe-tRNAPhe. Binding of two molecules of Phe-tRNAPhe to the 30S.poly(U) complex followed by the addition of 50S subunits resulted in the formation of (Phe)2-tRNAPhe in 75-90% of the reassociated 70S ribosomes. These results prove that isolated 30S subunits contain two physically distinct centers for the binding of specific aminoacyl- (or peptidyl-) tRNA. Addition of 50S subunits results in the formation of whole 70S ribosomes with usual donor and acceptor sites.     

A decreased aminoacyl-transfer-ribonucleic acid-binding capacity of 40S ribosomal subunits resulting from hypophysectomy of the rat

A technique that permitted the reversible dissociation of rat liver ribosomes was used to study the difference in protein-synthetic activity between liver ribosomes of normal and hypophysectomized rats. Ribosomal subunits of sedimentation coefficients 38S and 58S were produced from ferritin-free ribosomes by treatment with 0.8m-KCl at 30 degrees C. These recombined to give 76S monomers, which were as active as untreated ribosomes in incorporating phenylalanine in the presence of poly(U). Subunits from normal and hypophysectomized rats were recombined in all possible combinations and the ability of the hybrid ribosomes to catalyse polyphenylalanine synthesis was measured. The results show that the defect in ribosomes of hypophysectomized rats lies only in the small ribosomal subunit. The 40S but not the 60S subunit of rat liver ribosomes bound poly(U). The only requirement for the reaction was Mg(2+), the optimum concentration of which was 5mm. No apparent difference was seen between the poly(U)-binding abilities of 40S ribosomal subunits from normal or hypophysectomized rats. Phenylalanyl-tRNA was bound by 40S ribosomal subunits in the presence of poly(U) by either enzymic or non-enzymic reactions. Non-enzymic binding required a Mg(2+) concentration in excess of 5mm and increased linearly with increasing Mg(2+) concentrations up to 20mm. At a Mg(2+) concentration of 5mm, GTP and either a 40-70%-saturated-(NH(4))(2)SO(4) fraction of pH5.2 supernatant or partially purified aminotransferase I was necessary for binding of aminoacyl-tRNA. Hypophysectomy of rats resulted in a decreased binding of aminoacyl-tRNA by 40S ribosomal subunits.     

The protein-synthesizing activity of ribosomes isolated from the mammary gland of lactating and pregnant guinea pigs

1. Polyribosome preparations were made from the deoxycholate-treated post-nuclear fractions obtained by the disruption of mammary glands from lactating and pregnant guinea pigs. 2. A high proportion of large polyribosomes was obtained from the glands of lactating animals whereas mainly small polyribosomes were obtained from the glands of pregnant animals. The isolated preparations incorporated [(14)C]phenylalanine into protein. The polyribosomes from the glands of pregnant animals were less active than those from the glands of lactating animals but the activity of the former was stimulated more by poly(U) than was the latter. 3. The ribosomes from mammary gland could be dissociated into subunits after incubation, under conditions necessary for protein synthesis, in the presence of puromycin. The subunits could be recombined to give a preparation that actively polymerized [(14)C]phenylalanine in the presence of poly(U). The subunits from guinea-pig mammary gland could be combined with subunits from liver of either guinea pig or rat. Hybrid ribosomes were also formed from subunits derived from glands of pregnant and lactating animals. The hybrids were as active as were the ribosomes formed by reassociation of subunits from the same tissue, suggesting that in this respect the ribosomes from pregnant animals were not defective. 4. Polyribosomes from mammary glands of lactating animals when incubated with cell sap from the same source were tested for their ability to synthesize alpha-lactalbumin. The polyribosomes were incubated in the presence of [(3)H]leucine and alpha-lactalbumin was isolated from the supernatant. The protein was finally treated with cyanogen bromide and the C-terminal and N-terminal fragments were separated and their radioactivity was determined. Both fragments were radioactive consistent with the synthesis of alpha-lactalbumin. 5. The results are discussed in relation to protein synthesis in the mammary gland after parturition.     

Role of ribosomes in cycloheximide resistance of Neurospora mutants

Summary In Neurospora crassa, mutants resistant against cycloheximide appear with a marked time lag after mutation induction. We have suggested (Neuhäuser et al., 1970) that this lag indicates the time needed for the synthesis of altered ribosomes (phenotypic lag), that the drug in the wildtype acts upon the ribosomes, and that resistance is due to alterations in them.By measurements of poly-U directed poly-Phe synthesis on ribosomes of the wildtype and two different cycloheximide resistant mutants in a cell free system it is shown here that mutant ribosomes indeed differ from those of the wildtype. Poly-Phe synthesis on mutant ribosomes proceeds in the presence of the drug, whereas that on wildtype ribosomes is inhibited. This means that the earlier suggestions are correct.Abbreviation CHX cycloheximide     

Isolation and characterization of an inhibitor of ribosome-dependent GTP hydrolysis by elongation factor G

Two inhibitors of ribosome-dependent GTP hydrolysis by elongation factor (EF)G were found in the ribosome wash of Escherichia coli strain B. One of these inhibitors was purified to homogeneity and characterized. The isolated inhibitor was found to consist of two polypeptide subunits with apparent molecular masses of 23 kDa and 10 kDa. Inhibition of EF-G GTPase could not be overcome by increasing amounts of the elongation factor or high concentrations of GTP, but was reversed by large amounts of ribosomes. The effect of the inhibitor was reduced by increasing concentrations of either 30S or 50S ribosomal subunits. EF-G-dependent GTPase of 50S ribosomal subunits was not affected by the inhibitor. These findings clearly show that the inhibitor interferes with the modulation of EF-G GTPase activity by the interactions between 30S and 50S ribosomal subunits. Under conditions, where 30S CsCl core particles are able to associate with 50S subunits and to stimulate EF-G GTPase, the effect of the inhibitor was considerably reduced when intact 30S ribosomal subunits were substituted by 30S CsCl core particles. This finding indicates that 30S CsCl split proteins are important for the action of the inhibitor and that the inhibitor does not affect the EF-G GTPase merely by interfering with the association of ribosomal subunits. Furthermore, poly(U)-dependent poly(phenylalanine) synthesis was considerably less sensitive to the inhibitor than EF-G GTPase. When ribosomes were preincubated with poly(U) and Phe-tRNA(Phe), poly(phenylalanine) synthesis was considerably less affected by the inhibitor, whereas EF-G GTPase was still sensitive.     

The 60S ribosomal subunit is altered in the skeletal muscle of dystrophic hamsters

Polysomes from the skeletal muscle of normal and dystrophic hamsters were dissociated into ribosomal subunits by treatment with puromycin and the subunits from both strains were reassociated in all possible combinations. When their protein synthesis activity was assayed in a poly(U)-directed cell-free system at a low magnesium concentration, the reassociated ribosomes from dystrophic hamsters were less active than the ribosomes from control animals. The ribosomal defect is a property of the 60S subunit and is due to a ribosomal component rather than to abnormal binding of a non-ribosomal protein.     

Preparation of active run-off 80S ribosomes and their subunits from mouse liver.

A method is described for the preparation of active "run-off" 80S ribosomes and 40S and 60S subunits of mouse liver. A polysome preparation was incubated at 37 degrees C for 10 min under the condition for protein synthesis (4 mM Mg2+, 100 mM KCL). Puromycin (10 mM)and 2 M KCL were added to a final concentration of 0.1 mM and 500 mM, respectively, and the reaction mixture was further incubated at 37 degrees C for 10 min. This latter treatment destabilized small polysomes and "stuck" 80S particles, which were remaining after the first incubation, leading to complete release of 40S and 60S particles. Thus, the present method minimized variations in yield of subunits due to polysome preparations and preincubation conditions. The subunits were separated by sucrose density-gradient centrifugation or recovered by precipitation following reassociation into 80S particles (run-off 80S). The reformation of 80S particles from the subunits occurred spontaneously at 5 mM Mg2+ and 100mM KCL. The isolated 40S and 60S subunits, separately, showed low phenylalanine-incorporating activity in the presence of poly(U), but when recombined, polymerized up to 10 phenylalanine residues per couple.