Temperature sensitivity of protein synthesis initiation. Inactivation of a ribosomal factor by an inhibitor formed at elevated temperatures.

When a reticulocyte lysate, supplemented with hemin, was warmed at 42 °C, its protein-synthesizing activity was greatly decreased. This was accompanied by the reduced formation of the 40 S·Met-tRNA_f initiation complex. This complex preformed at 34 °C, however, was stable and combined with added globin mRNA and the 60 S ribosomal subunit to form the 80 S complex at the elevated temperature. When the ribosome-free supernatant fraction of lysates was warmed at 42 °C with hemin and then added to the fresh lysate system, it inhibited protein synthesis by decreasing the formation of the 40 S complex. This decrease in protein synthesis by warmed lysates or warmed supernatant could be overcome by high concentrations of GTP and cyclic AMP. This effect of GTP and cyclic AMP was antagonized by ATP. The results indicate that the inactivation of protein synthesis by the lysate warmed at 42 °C is due to the formation of an inhibitor in the supernatant. The ribosomal KCl extract prepared from the lysate that had been warmed at 34 °C and then incubated at this temperature for protein synthesis supported protein synthesis by the KCl-washed ribosome at both 34 and 42 °C. On the contrary, the extract from lysates that had been warmed at 42 °C and then incubated at 34 °C could not support protein synthesis at 42 °C, although it was almost equally as promotive as the control extract in supporting protein synthesis at 34 °C. The results indicate that the factor which can protect protein synthesis against inactivation at 42 °C is itself inactivated in lysates warmed at 42 °C. However, the activity of this extract to support formation of the ternary complex with Met-tRNA_f and GTP was not reduced. Native 40 S ribosomal subunits isolated from lysates that had been warmed at 42 °C and then incubated for protein synthesis indicated that the quantity of subunits of density 1.40 g/cm³ in a CsCl density gradient were decreased while those of density 1.49 g/cm³ were increased. The factor-promoted binding of Met-tRNA_f to the 40 S subunit of lower density from the warmed and unwarmed lysates was equal, suggesting that the ribosomal subunit was not inactivated. These results were discussed in terms of the action of the inhibitor formed in the supernatant at 42 °C, which may inactivate a ribosomal factor essential for protein synthesis initiation.     

Temperature sensitivity of protein synthesis initiation in reticulocyte cell-free systems. A ribosomal factor which protects protein synthesis inactivation at elevated temperatures.

Inactivation of protein synthesis in the reticulocyte lysate system, which occurs when the system is incubated at 42 °C, was prevented by a high concentration KCl extract of the ribosomes. The KCl extract also supported protein synthesis at 42 °C by KCl-washed ribosomes. Three factor fractions (IF.15, IF.2, and IF.25) were separated from the extract and characterized in partial reactions of initiation. The factor IF.2 could prevent the inactivation of the factor IF.15-promoted protein synthesis by the washed ribosomes at 42 °C. IF.2 also overcame the decrease in IF.15-promoted 40S subunit-Met-tRNA_f complex at 42 °C. The protective activity of IF.2 was inactivated by N-ethylmaleimide. The activities of IF.15 and IF.2 were little affected by heating the factors at 42 °C. However, prewarming of KCl-washed ribosomes at 42 °C caused decreased protein synthesis in subsequent incubation at 34 °C with unwarmed factors. These results suggest that some components other than the initiation factors may be inactivated at 42 °C, which is prevented by IF.2 in the course of protein synthesis.     

Regulation of protein synthesis in heat-shocked Drosophila cells. Soluble factors control translation in vitro

We have developed an in vitro translation system from heat-shocked and normal Drosophila cultured cells. The lysates retain regulation of translation typical of the whole cells from which they were prepared, both when programmed by endogenous mRNA and when RNA-dependent. These systems have been used to investigate the mechanism of shutdown of normal protein synthesis and selection of heat shock mRNAs for translation in heat shock in Drosophila. Supplementation of intact RNA-dependent lysates with separated ribosome or supernatant fractions from normal or heat-shocked translation systems showed the normal supernatant fraction could "rescue" normal protein synthesis in a heat shock lysate. Normal ribosomes had no rescuing activity and neither heat shock fraction affected translation in normal lysates. Reconstitution of the system from separated ribosomes and supernatant in normal and mixed combinations showed heat shock and normal ribosomes were both competent to support normal protein synthesis with normal supernatant. Heat shock supernatant did not support normal protein synthesis with ribosomes from either source. We conclude that the factors regulating translation in heat-shocked Drosophila cells are soluble factors in the lysate and that the soluble factors present in the normal lysate are dominant.     

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.     

Subcellular localization of a lesion in protein synthesis in rabbit reticulocytes incubated at elevated temperatures.

When rabbit reticulocytes are incubated at 43-45 degrees C their rate of protein synthesis rapidly decreases, compared to a contol 37 degrees C incubation. Lysates prepared from cells incubated at this supra-optimal temperature have an equally decreased capacity for endogenous, but not poly(uridylic acid)-directed, protein synthesis. Subcellular fractionation traced the lesion to the crude ribosomal pellet, 0.5 M KCl ribosomal wash and postribosomal supernatant of the temperature-shocked cells. Preparation of purified ribosomal subparticles showed, however, that they were as active as the control in protein synthesis. In this paper we present evidence that the decreased activity of the heated lysate, 0.5 mM KCl wash and postribosomal supernatant is due to an inhibitor and can be overcome by the addition of 0.5 M KCl or supernatant from control cells. The results are discussed in terms of the inactivation of a component, essential for initiation of endogenous protein synthesis, which is probably partitioned between ribosomes and supernatant. We also suggest that the decreased protein synthetic activity of the heated cells may be related to their decreased synthesis of haem.     

ISOLATION OF TWO DISTINCT CLASSES OF POLYSOMES FROM A NUCLEAR FRACTION OF RAT LIVER

Isolated rat liver nuclei were washed with Triton-X-100 in the presence of liver cell sap. This treatment liberated a fraction of polysomes which were isolated by differential centrifugation and were designated "outer membrane polysomes." The outer membrane polysomes synthesized protein in vivo. Shortly after injection of orotic acid-¹⁴C, the RNA of outer membrane polysomes had a higher specific activity than that of cytoplasmic polysomes. It was postulated that outer membrane polysomes may be an intermediate in the transfer of newly synthesized RNA from the nucleus to the cytoplasm. In other experiments, Triton-washed rat liver nuclei were lysed in the presence of deoxycholate and deoxyribonuclease. A ribonucleoprotein fraction was isolated from the lysate by differential centrifugation. This fraction contained "intranuclear ribosomes," which sedimented like partially degraded polysomes in sucrose gradients. This degradation could be partially prevented if intranuclear ribosomes were purified by sedimentation through heavy sucrose. The resulting pellets were termed "intranuclear polysomes" because they contained some undergraded polysomes. Intranuclear polysomes were highly radioactive after a brief pulse with orotic acid-¹⁴C, but did not appear to synthesize protein rapidly in vivo. Intranuclear polysomes may represent the initial stage of assembly of polyribosomes in the nucleus.     

ON THE MECHANISM OF ELECTROSHOCK-INDUCED INHIBITION OF PROTEIN SYNTHESIS IN RABBIT CEREBRAL CORTEX

Abstract— The mechanism of electroshock (ES)-induced inhibition of protein synthesis in rabbit cerebral cortex has been investigated by using a cell-free system. The protein biosynthetic activity of the post-mitochondrial supernatant (PMS) obtained from the cerebral cortex of ES-treated animals was found to be markedly lower than in controls (C). This inhibition was accompanied by a decrease of polysomes and an increase of monomers. In addition, a relative increase in light polysomes was evident at short intervals after ES treatment. No difference was found in the total soluble activity and in the activity of the elongation factors and ribonuclease present in the cell sap of C and ES animals. The biosynthetic activity of ES-total. free and membrane-bound ribosomes was approx 45% lower than that of the corresponding C fractions: polysome/monomer ratios were similarly reduced. The total content of cortical ribosomes was not affected by ES. Following ES treatment there was no change in the ribo-somal ability to elongate, terminate and release polypeptide chains, nor a decrease in the polysomal content of poly(A)-containing mRNA. These data strongly suggest that the ES-induced inhibition of protein synthesis results from a defect in the initiation process. The possible mechanisms mediating this defect have been discussed.     

A ribosome-free extract from cultured cells improves recovery of polysomes from the mosquito fat body: analysis of vitellogenin and ribosomal protein rpL34 gene expression

We have examined the association of ribosomal protein rpL34 mRNA with polysomes in Aedes albopictus C7-10 cells in culture using a simple, two-step sucrose gradient. In growing cells, 40-50% of the ribosomes were engaged on polysomes. This proportion could be increased to 80% when metabolism was stimulated by refeeding the cells with fresh medium. Conversely, ribosomes shifted off polysomes when cells were starved with phosphate-buffered saline or cell lysates were treated with puromycin. When similar approaches were used with fat body from blood-fed female Aedes aegypti mosquitoes, we were unable to obtain the polysome fraction that contained vitellogenin mRNA, which is abundantly translated after a blood meal. Addition of post-mitochondrial supernatant from fat body to polysomes from cultured cells shifted the polysome profile towards smaller polysomes and monosomes, in a dose-dependent fashion. Disruption of fat body tissue in a post-ribosomal supernatant from refed cells improved the recovery of polysomes, demonstrating both the engagement of vitellogenin mRNA on polysomes and the mobilization of rpL34 from messenger-ribonuceloprotein particles onto polysomes in blood-fed mosquitoes. These observations suggested that ribonucleases remain active when polysomes are prepared from mosquito fat body, and that cell culture supernatant contains a ribonuclease inhibitor.     

Neurospora crassa temperature-sensitive mutant apparently defective in protein synthesis.

A temperature-sensitive mutant of Neurospora was isolated which appeared to be defective in the initiation of protein synthesis. The defect in mutant 34Cts was apparently due to a single gene mutation, and was recessive in heterokaryons. Conidial germination was normal and hyphal growth was nearly so in the mutant at 20 C, but both were greatly inhibited at 35 C. After 15 min at 35 C there was a reduced rate of protein synthesis, followed by decreases in ribonucleic acid and deoxyribonucleic acid synthesis. The percentage of ribosomes in polysomes declined at 35 C and the average size of polysomes decreased. Because the decrease in protein synthesis, it was believed that some part of the translational system may be affected by the mutation. Mutant 34Cts was given the designation psi-1.     

Isolation and characterization of ribosomes from thoraces of the sheep blowfly,Lucilia cuprina,and their capacity for protein synthesis at different stages of development

The isolation and characterization of ribosomal fractions from thoraces of Lucilia cuprina have been described and their capacity for in vitro protein synthesis studied at different stages of development. There are large differences in the yield of ribosomes during development, and these probably reflect differences in the number of ribosomes in vivo. The amino acid incorporation activity varied at different stages of development and this variation correlated with the percentage of polysomes present. Maximal activity was found just after adult emergence. Results obtained by the use of supernatant fractions from different stages of development suggest that control of protein synthesis in this insect takes place at the level of translation.     

Inactivation of eucaryotic ribosomes by the toxic plant proteins abrin and ricin

The effect of abrin and ricin on protein synthesizing systems from different sources was studied. The protein synthesis in a cell-free system from rabbit reticulocytes and from rat liver was strongly inhibited by the toxins, whereas a system from E. coli was not affected. Separate treatments of ribosomes and postribosomal supernatant from a rabbit reticulocyte lysate showed that the site of action of the toxins is on the ribosomes. The inactivation of the rabbit reticulocyte lysate by the toxins was a function of the incubation time and temperature. Protein synthesis was not necessary for the toxins to exert their effect. The data indicate that abrin and ricin act only on the eucaryotic type of ribosomes, and that they exert their effect by enzymatic action.     

Incomplete polypeptides are formed in vitro by premature chain termination

The mechanism of incomplete polypeptides formation during protein synthesis was studied in the wheat germ cell-free system programmed with brome mosaic virus RNA 4. The synthesis of coat protein, the complete product of RNA 4 translation, was accompanied by the appearance of polypeptides of lower molecular mass. It was shown that incomplete products are formed by translation of different lengths of RNA 4, always from the first 5' AUG codon, and were due neither to proteolysis of coat protein nor to the translation of nucleolytic fragments of mRNA. The molecular masses of incomplete products were determined and the nucleotide sequence of RNA 4 was examined in the regions where wheat germ ribosomes stop translating. It was found that they contained, on average, a slightly higher guanosine content than the total coding part of RNA 4. Translation of RNA 4 in the reticulocyte lysate resulted in a marked diminution of incomplete polypeptides. Addition of high-speed supernatant from reticulocyte lysate prevented the formation of incomplete products during translation of RNA 4 in the wheat germ system. This suggests that reticulocyte lysate contains some factor(s) which facilitate the movement of ribosomes beyond the regions where the elongation is retarded.     

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.     

Regulation of translation in rabbit reticulocytes and mouse L-cells; comparison of the effects of temperature

Various parameters of protein synthesis were analyzed in rabbit reticulocytes exposed to various temperatures for up to five hours. Between 10°C and 40°C total protein synthesis exhibited two different apparent activation energies (36 kcal/mole, 10–24°C; 22 kcal/mole, 24–40°C), as did protein elongation and release (35 kcal/mole, 10–25°C; 12 kcal/mole, 25–40°C). However, the level of polysomes remained essentially unchanged between 0°C and 42°C which implies that the activation energy for polypeptide initiation is quite similar to that for elongation and is also biphasic. This situation is different from that in cultured mouse L-cells where the polysome level is dependent on temperatures. Nevertheless, reticulocytes and L-cells appear to be similar in their temperature dependence of initiation and in their rate of elongation (5–6 amino acids/second at 36°C).     

Evidence for the presence of an inhibitor on ribosomes in mouse L cells infected with mengovirus.

After infection of mouse L cells with mengovirus, there is a rapid inhibition of protein synthesis, a concurrent disaggregation of polysomes, and an accumulation of 80S ribosomes. These 80S ribosomes could not be chased back into polysomes under an elongation block. The infected-cell 80S-ribosome fraction contained twice as much initiator methionyl-tRNA and mRNA as the analogous fraction from uninfected cells. Since the proportion of 80S ribosomes that were resistant to pronase digestion also increased after infection, these data suggest that the accumulated 80S ribosomes may be in the form of initiation complexes. The specific protein synthetic activity of polysomal ribosomes also decreased with time of infection. However, the transit times in mock-infected and infected cells remained the same. Cell-free translation systems from infected cells reflected the decreased protein synthetic activity of intact cells. The addition of reticulocyte initiation factors to such systems failed to relieve the inhibition. Fractionation of the infected-cell lysate revealed that the ribosomes were the predominant target affected. Washing the infected-cell ribosomes with 0.5 M KCI restored their translational activity. In turn, the salt wash from infected-cell ribosomes inhibited translation in lysates from mock-infected cells. The inhibitor in the ribosomal salt wash was temperature sensitive and micrococcal nuclease resistant. A model is proposed wherein virus infection activates (or induces the synthesis of) an inhibitor that binds to ribosomes and stops translation after the formation of the 80S-ribosome initiation complex but before elongation. The presence of such an inhibitor on ribosomes could prevent them from being remobilized into polysomes in the presence of an inhibitor of polypeptide elongation.     

Inhibition of peptide chain initiation by a nonhemin-regulated translational repressor from Friend leukemia cells.

A nonhemin-regulated translational repressor protein has been purified partially from the postribosomal supernatant fraction of Friend leukemia cells grown in the absence of dimethylsulfoxide. This repressor inhibits protein synthesis in lysates from rabbit reticulocytes or Friend leukemia cells and in a fractionated system using Artemia salina ribosomes, reticulocyte mRNA, and soluble components from reticulocytes. In contrast, the hemin-controlled repressor from reticulocytes does not inhibit protein synthesis in lysates from Friend leukemia cells. The repressor from Friend leukemia cells has no effect on poly(U)-directed synthesis of polyphenylalanine using reticulocyte ribosomes nor on the extension and release of nascent globin chains that were initiated in intact reticulocytes. It does not block completion of peptides on ribosomes isolated from reticulocytes incubated with NaF nor does it inhibit initiation factor-dependent formation of methionylpuromycin, but it inhibits globin mRNA-dependent methionylvaline synthesis. The Friend leukemia cell repressor promotes peptide synthesis-dependent breakdown of polysomes in reticulocyte lysates that appears to involve inhibition of ribosome reattachment to mRNA during peptide chain initiation. It is concluded that the Friend leukemia cell repressor blocks peptide initiation at a point between the addition of methionyl-tRNA^fMet to the ribosomal initiation complex and the NaF-sensitive reaction.     

DNA sequences flanking the starts of the hsp 70 and alpha beta heat shock genes are homologous

To determine whether ribosomes have a role in the postfertilization activation of protein synthesis in sea urchin eggs, we measured the translational activity of ribosomes isolated from unfertilized eggs and embryos of Strongylocentrotus purpuratus. Numerous previous studies have indicated few if any differences in the activity of such ribosomes. However, by using improved physiological isolation and in vitro conditions, we have found important differences in the activities of egg and embryo ribosomes. Ribosomes obtained from blastula polyribosomes were active in translating reticulocyte mRNA in a ribosome-dependent cell-free translation system, whereas ribosomes obtained from unfertilized eggs became fully active only after a characteristic, reproducible delay of up to 15 min at 26°C. The extent of this delay varied with incubation pH, but not with concentrations of K⁺, Mg²⁺, initiation factors, or mRNA. However, at incubation pH between 6.90 and 7.65, the egg ribosomes were always less active than blastula ribosomes.     

Comparison of cell-free protein synthesis by different regions of chicken brain

The cell-free protein synthesis by the postmitochondrial supernatant from chicken cerebrum was twofold greater than protein synthesis by the cerebellum or optic lobes. Ribosomal aggregation of mRNA and ribonuclease activity of the postmitochondrial supernatant from the three brain regions was not statistically different. The higher protein synthetic activity of the cerebral postmitochondrial supernatant was associated with both the postribosomal supernatant (cell sap) and microsomal fractions. Cerebral monomeric ribosomes were more active in polyuridylic acid directed polyphenylalanine synthesis than monomeric ribosomes from either the cerebellum or optic lobes. The ability of cerebral cell sap to support polyuridylic acid directed polyphenylalanine synthesis was 1.6 to 2 times greater than cell sap from the other two regions. Cell sap factors other than tRNA^phe or phenylalanyl-tRNA synthetases appear to be responsible for the higher protein synthetic activity of the cbr cell sap.     

Ribosome changes during translation

Studies on the quantitative binding of [³H]anisomycin are useful in determining conformational and/or structural changes on eukaryotic ribosomes. We have shown that yeast ribosomes have different structures depending on their functional states during the ribosome cycle as defined by their affinity for [³H]anisomycin.Free ribosomes, either in vivo run-off ribosomes (1 mm-sodium azide treatment or 8 °C incubation of spheroplasts) or puromycin-dependent released ribosomes, have an affinity defined by K_d = 3.3 to 3.6 μm.Ribosomes forming polysomes engaged in protein synthesis have at least two new different conformations (defined by K_d,H = 0.81 μm and K_d,L = 12 μm). These conformations have been ascribed to the pre and post-translocated steps of the elongation cycle in protein synthesis by blocking the polysomes with specific inhibitors of translation. Pre-translocated polysomes (polysomes blocked with cycloheximide) have an affinity of K_d^CHX = 12 μm and post-translocated polysomes (polysomes blocked with doxycycline) have an affinity of K_d^DC = 0.82 μm. These dissociation constants are identical to K_d,L and K_d,H obtained with control untreated polysomes, respectively.Moreover, a new ribosome conformation defined by K_d^DT = 1.5 μm and K_d^FA = 1.8 μm was found, by blocking the polysomes with the elongation factor, EF-2, bound by using either diphtheria toxin or fusidic acid.We also present evidence of the previously reported heterogeneity of standard preparations of eukaryotic ribosomes (Barbacid & Vazquez, 1974a) being a direct consequence of the high-salt washing treatment of ribosomes.     

Inhibition of protein synthesis by double-stranded RNA in reticulocyte lysates: evidence for activation of an endoribonuclease.

Double-stranded RNA is a potent inhibitor of protein synthesis in rabbit reticulocyte lysates. Three lines of evidence suggest that at least part of this inhibitory activity is due to activation of a nuclease which degrades mRNA: (1) In the presence of emetine reticulocyte polysomes are partially degraded to structures containing 1–3 ribosomes; (2) 34S Mengo-virus RNA is degraded to fragments sedimenting at less than 18S; (3) The template activity of globin mRNA extracted from the lysates is reduced by 90% when compared to appropriate controls. The ability of double-stranded RNA to activate a nuclease in the reticulocyte system is very similar to that observed in extracts from interferon treated cells and probably involves formation of the unusual oligonucleotide pppA^2′ p^5′ A^2′ p^5′ A.