Protein Synthesis In Extracts Research Articles

Protein kinase R and the inflammasome.

Protein kinase R (PKR) was first identified as a mediator of the double-stranded RNA (dsRNA)-mediated inhibition of protein synthesis in extracts from interferon-treated cells. In a physiological context, viral replication results in production of dsRNA, activation of PKR by autophosphorylation, and phosphorylation of the protein synthesis initiation factor eIF2α. Subsequent biochemical, structural, and genetic analyses have identified the dsRNA and kinase domain structure of PKR, and shown that its deletion from the germline of mice results in impaired resistance to infection by many different viruses. These studies have also opened up roles for PKR in different signaling pathways, the most recent being regulation of the inflammasome. Here we review evidence for this newly ascribed function for PKR and discuss roles in inflammasome regulation and associated diseases.

An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation

In vitro translation systems are used to investigate translational mechanisms and to synthesize proteins for characterization. Most available mammalian cell-free systems have reduced efficiency due to decreased translation initiation caused by phosphorylation of the initiation factor eIF2alpha on Ser51. We describe here a novel cell-free protein synthesis system using extracts from cultured mouse embryonic fibroblasts that are homozygous for the Ser51 to- Ala mutation in eIF2alpha (A/A cells). The translation efficiency of a capped and polyadenylated firefly luciferase mRNA in A/A cell extracts was 30-fold higher than in wild-type extracts. Protein synthesis in extracts from A/A cells was active for at least 2 h and generated up to 20 microg/mL of luciferase protein. Additionally, the A/A cell-free system faithfully recapitulated the selectivity of in vivo translation for mRNA features; translation was stimulated by a 5'-end cap (m7GpppN) and a 3'-end poly(A) tail in a synergistic manner. The system also showed similar efficiencies of cap-dependent and IRES-mediated translation (EMCV IRES). Significantly, the A/A cell-free system supported the post-translational modification of proteins, as shown by glycosylation of the HIV type-1 gp120 and cleavage of the signal peptide from beta-lactamase. We propose that cell-free systems from A/A cells can be a useful tool for investigating mechanisms of mammalian mRNA translation and for the production of recombinant proteins for molecular studies. In addition, cell-free systems from differentiated cells with the Ser51Ala mutation should provide a means for investigating cell type-specific features of protein synthesis.

Inhibition of bacterial protein synthesis by elongation-factor-Tu-binding antibiotics MDL 62,879 and efrotomycin.

MDL 62,879 (formerly GE 2270 A) is a novel antibiotic active against Gram-positive bacteria by inhibiting protein synthesis. MDL 62,879 is not active against Gram-negative bacteria, but inhibits cell-free protein synthesis in extracts from Escherichia coli, and shows a high binding affinity for its elongation factor Tu (EF-Tu). We prepared ribosomes and protein-synthesis elongation factors from three sources: E. coli, Bacillus subtilis, and a strain of B. subtilis selected for resistance to MDL 62,879 (strain G1674). Homologous and heterologous reconstituted systems were used to compare the effects of MDL 62,879 and of efrotomycin, an EF-Tu inhibitor of the kirromycin class, which is inactive against both B. subtilis and E. coli. We showed that in cell-free protein synthesis: (a) E. coli was sensitive to both MDL 62,879 and efrotomycin; (b) B. subtilis was sensitive to MDL 62,879, but not to efrotomycin; (c) B. subtilis G1674 was resistant to both antibiotics. In the E. coli system and in the system from wild-type B. subtilis, inhibition by MDL 62,879 was reversed upon addition of purified EF-Tu from B. subtilis G1674. This demonstrates that the antibiotic acts by inhibition of EF-Tu. In contrast, extracts from B. subtilis failed to restore activity in an efrotomycin-inhibited E. coli system. Dominance or resistance to MDL 62,879 and of sensitivity to efrotomycin in heterologous cell-free protein synthesis confirms that inhibition of EF-Tu by the two antibiotics is mediated by different mechanisms of action.

The effect of temperature shifts on protein synthesis by the psychrophilic bacterium Vibrio sp. strain ANT-300

In the psychrophilic bacterium Vibrio sp. strain ANT-300, the temperature-related characteristics of protein synthesis in cells grown at 0 degrees C differed from those of cells grown at 13 degrees C. Cells grown at 0 degrees C and 13 degrees C transported amino acids at the same rates, dependent on the temperature at which rates were measured. The rates of protein synthesis in extracts of cells grown at 0 degrees C and at 13 degrees C differed, as a result of the changes in the properties of the soluble fraction involved in protein synthesis. Concurrently, levels of more than 24 polypeptides in the soluble fraction changed considerably. These results suggest that the difference in temperature dependence of protein synthesis in cells grown at various temperatures may be brought about by specific changes in the levels of a small number of polypeptides (less than 15% of the total number of proteins detected by silver-staining) in response to a change in temperature.

Binding of novel macrolide structures to macrolides-lincosamides-streptogramin B-resistant ribosomes inhibits protein synthesis and bacterial growth

Dimethylation of adenine 2058 in 23S rRNA renders bacteria resistant to macrolides, lincosamides, and streptogramin B (MLS resistance), because the antibiotic binding site on the altered 50S ribosomal subunit is no longer accessible. We now report that certain 6-O-methyl-11,12-cyclic carbamate derivatives of erythromycin are able to bind to dimethylated MLS-resistant 50S ribosomal subunits, thus inhibiting protein synthesis and cell growth. One of these novel structures, an 11-deoxy-11-(carboxyamino)-6-O-methylerythromycin A 11,12-(cyclic ester) derivative, structure 1a, was studied in detail. It inhibited in vitro protein synthesis in extracts prepared from both susceptible and MLS-resistant Bacillus subtilis with 50% inhibitory concentrations of 0.4 and 20 microM, respectively. The derivative bound specifically to a single site on the 50S subunit of MLS-resistant ribosomes prepared from B. subtilis and Staphylococcus aureus, and no binding to 30S subunits was observed. The association rate constant of derivative 1a with sensitive and resistant ribosomes was 100- and 500-fold slower, respectively, than that of the parent compound, erythromycin, with sensitive ribosomes. The dissociation rate constant of 1a from sensitive and resistant ribosomes was 50- to 100-fold slower than the rate of erythromycin dissociation from sensitive ribosomes. Furthermore, 1a binding to sensitive 50S subunits led to induction of ermC and ermD, while binding to resistant 50S subunits did not, showing that perturbation of sensitive and resistant 50S subunit function by 1a differs. These data demonstrated that 1a is unique in its interaction with MLS-resistant ribosomes and that this interaction causes a novel allosteric perturbation of ribosome function.

Decreased accuracy of protein synthesis in extracts from aging human diploid fibroblasts

The accuracy of protein synthesis has been measured in extracts from human diploid fibroblasts of different ages. Extracts were supplied with purified mRNA for the coat protein of the cowpea variant of tobacco mosaic virus (CcTMV), which lacks condons for cysteine and methionine. The presence of 35S-cysteine in CcTMV coat protein synthesized during translation reactions therefore represents translational error. Translation reactions were performed with extracts from young fibroblasts (less than 50% of life span completed) and old fibroblasts (more than 90% of life span completed), and the translation products were purified by immunoprecipitation and analyzed by polyacrylamide gel electrophoresis. The error frequency increased from 4.2 × 10 −5 cysteines/amino acid in young cell extracts to 2.9 × 10 −4 cysteines/amino acid in old cell extracts. Cysteine incorporation was not due to nonspecific binding, and could be increased approximately sixfold by the addition of the misreading antibiotic, paromomycin. It is concluded that translational accuracy is not stable during aging in vitro, and it is proposed that this decrease in the fidelity of information transfer could be responsible for the variety of changes observed in aging cultured human cells.

Effects of temperature upon the cell-free translation system from Coxiella burnetii.

The rate and extent of coliphage Q beta RNA translation by cell-free extracts prepared from Coxiella burnetii were studied. When translations were conducted at temperatures elevated above 37 degrees C, both polypeptide elongation and frequency of initiation were by comparison increased. The ratios of products synthesized from the polycistronic phage mRNA also changed upon increases in translation temperature, especially at 45 degrees C. Although the organism is a moderate acidophile, initiation of protein synthesis in extracts did not occur below pH 6.2, and was superior when the pH was 6.8-7.2. The results are discussed in context with the known physiological characteristics of this obligate intracellular bacterium.

Circadian changes in protein-synthesis rate and protein phosphorylation in cell-free extracts of Gonyaulax polyedra

The polysomal pattern of the dinoflagellate Gonyaulax polyedra, cultured under constant conditions, demonstrates a circadian rhythm. The relative amount of polysomes increases during the phase corresponding to the previous night period (=subjective night phase) when the rate of protein synthesis reaches its maximum (Cornelius et al., 1985, Planta 160, 365-370). Cell-free extracts were isolated at different circadian phase. The rate of protein synthesis in the extracts changed rhythmically in the same manner as the rate of protein synthesis in vivo. Substances in the postribosomal supernatants influenced the protein-synthesis rate of the cell-free system, depending on the phase when they were isolated: "night factors" stimulated protein synthesis in "day extracts" whereas "day factors" inhibited protein synthesis in "night extracts". These effects were abolished by heating the postribosomal supernatant. In-vitro phosphorylation in parallel probes showed changes in the pattern of phosphorylated proteins. Phosphorylation of one of the proteins (95 kDa) was decreased after addition of "night factor(s)" and increased after addition of "day factor(s)". Cyclic-AMP enhanced the rates of protein synthesis and phosphorylation in the day extracts.

Streptococcal tetracycline resistance mediated at the level of protein synthesis.

The mechanism of tetracycline resistance was examined in strains containing each of the three previously identified resistance determinants in Streptococcus spp. Uptake of tetracycline was measured in tetracycline-sensitive cells as well as in cells containing each of the three resistance determinants. In cells containing tetL, uptake was not observed. However, in sensitive cells and cells containing either tetM or tetN, tetracycline was accumulated approximately 25-fold against a concentration gradient. Furthermore, there was no evidence for modification of intracellular tetracycline recovered from sensitive, tetM, or tetN cells. Protein synthesis in extracts derived from organisms containing tetM or tetN was resistant to tetracycline. In contrast, extracts of sensitive and tetL cells were sensitive to tetracycline.

Protein synthesis in lysates of Aedes albopictus cells infected with vesicular stomatitis virus.

Aedes albopictus cells (clone LT-C7) showed a marked cytopathic effect and inhibition of protein synthesis (both host and viral) after infection with vesicular stomatitis virus (VSV), but only if (i) cultures were incubated at 34 degrees C rather than 28 degrees C and (ii) serum was present in the medium (S. Gillies and V. Stollar, Mol. Cell. Biol. 2:66-75, 1982). To learn more about how protein synthesis is shut off in VSV-infected A. albopictus cells, we have compared cell-free protein synthesis in extracts prepared from VSV-infected cells and control cells. Extracts prepared 6 h after infection from VSV-infected cells maintained at 34 degrees C in the presence of serum reflected what was observed with intact cells in at least two respects: (i) they showed a markedly diminished capacity to carry out protein synthesis (whether directed by endogenous or exogenously added mRNA), and (ii) there was decreased phosphorylation in vitro by [gamma-32P]ATP of a specific ribosomal protein (Gillies and Stollar, Mol. Cell. Biol. 2:66-75, 1982). In addition, and consistent with a block at the level of initiation, the formation of 80S initiation complexes, as measured by binding of VSV 12 to 18S mRNA, was reduced in the inactive extracts. Addition of an S-100 fraction from uninfected cells to the inactive extract reversed each of the aforementioned changes; i.e., it restored protein synthetic activity, it stimulated the formation of 80S initiation complexes, and it increased phosphorylation of the specific ribosomal protein referred to above. The active component in the S-100 fraction was heat labile and non-dialyzable and, upon ammonium sulfate fractionation of the S-100 fraction, was found in the 40 to 70% saturation fraction. Our findings suggest that VSV infection of A. albopictus cells inhibits protein synthesis by inactivating a macromolecular component, probably a protein, in the S-100 fraction which may be involved in the initiation of protein synthesis. More specifically, we suggest that this component is involved in the joining of the ribosomal subunits to form 80S initiation complexes.

Protein synthesis in lysates of Aedes albopictus cells infected with vesicular stomatitis virus.

Aedes albopictus cells (clone LT-C7) showed a marked cytopathic effect and inhibition of protein synthesis (both host and viral) after infection with vesicular stomatitis virus (VSV), but only if (i) cultures were incubated at 34 degrees C rather than 28 degrees C and (ii) serum was present in the medium (S. Gillies and V. Stollar, Mol. Cell. Biol. 2:66-75, 1982). To learn more about how protein synthesis is shut off in VSV-infected A. albopictus cells, we have compared cell-free protein synthesis in extracts prepared from VSV-infected cells and control cells. Extracts prepared 6 h after infection from VSV-infected cells maintained at 34 degrees C in the presence of serum reflected what was observed with intact cells in at least two respects: (i) they showed a markedly diminished capacity to carry out protein synthesis (whether directed by endogenous or exogenously added mRNA), and (ii) there was decreased phosphorylation in vitro by [gamma-32P]ATP of a specific ribosomal protein (Gillies and Stollar, Mol. Cell. Biol. 2:66-75, 1982). In addition, and consistent with a block at the level of initiation, the formation of 80S initiation complexes, as measured by binding of VSV 12 to 18S mRNA, was reduced in the inactive extracts. Addition of an S-100 fraction from uninfected cells to the inactive extract reversed each of the aforementioned changes; i.e., it restored protein synthetic activity, it stimulated the formation of 80S initiation complexes, and it increased phosphorylation of the specific ribosomal protein referred to above. The active component in the S-100 fraction was heat labile and non-dialyzable and, upon ammonium sulfate fractionation of the S-100 fraction, was found in the 40 to 70% saturation fraction. Our findings suggest that VSV infection of A. albopictus cells inhibits protein synthesis by inactivating a macromolecular component, probably a protein, in the S-100 fraction which may be involved in the initiation of protein synthesis. More specifically, we suggest that this component is involved in the joining of the ribosomal subunits to form 80S initiation complexes.

Protein synthesis in extracts from interferon-treated Mengo virus infected cells

We previously showed in intact L cells that interferon treatment did not modify the shut-off of cellular RNA and protein synthesis induced by infection with Mengo virus although viral replication is inhibited (1,2). We have also demonstrated that inhibition of host protein synthesis was not due to degradation of messengers since cellular mRNA could be extracted from interferon-treated infected cells and efficiently translated in a reticulocyte lysate(2). Cellular mRNA was not degraded although 2–5A was present as reported here. We prepared cell-free systems from such cells at a time when cellular shut-off was fully established. The undegraded messengers remained untranslated under cell-free protein synthesis conditions and almost no polysomes were detected. The decreased amount of [ 35S]Met-tRNA-40S complex observed in these lysates might account for the inhibition of protein synthesis at the level of initiation.

Effect of rate-limiting elongation on bacteriophage MS2 RNA-directed protein synthesis in extracts of Escherichia coli

The consequences of limiting the rate of elongation of protein synthesis in vitro have been examined. The concentration of Trp-tRNA Trp was manipulated by varying the amount of exogenously added tryptophan in extracts from an Escherichia coli mutant in which the tryptophanyl-tRNA-synthetase has a higher K M for tryptophan. The evidence presented supports the hypothesis that variation of the rate of elongation can be a means of regulating gene expression, both directly, by slowing or accelerating the rate of protein synthesis and indirectly, by leading to varying three-dimensional structures of the messenger RNA when progress of the ribosomes is perturbed. The data can be described by assuming that if a specific transfer RNA is limiting, to a first approximation the overall rate of protein synthesis is determined by the relative rate of reading past an individual codon requiring that tRNA raised to the power of how many times that codon appears in the message. This could be explained by a model in which, with a significant probability, the ribosome stops protein synthesis prematurely at these codons, falls off the messenger RNA and is available for further rounds of protein synthesis. In agreement with other work, evidence is also presented that suggests that under the most drastic available limitation of the elongation rate, that is, starvation for a given amino acid, reading through the corresponding “hungry codon” occurs in vitro at a surprisingly high rate, possibly due to mistranslation.

Biochemical and genetic evidence for a new class of emetine-resistant Chinese hamster cells with alterations in the protein biosynthetic machinery

We have isolated a number of emetine-resistant mutants from several different clones of CHO and CHL cells. Protein synthesis in extracts derived from each of the mutants is much more resistant to emetine than in the parental, emetine-sensitive cell lines, indicating the lesions affect the protein synthetic machinery directly. However, hybrid cell lines, derived from fusing either of two different emetine-resistant CHO mutants with either one of two different emetine-resistant CHL cells, are much more sensitive to growth inhibition by emetine than either parent. In addition, the incorporation of [3H] amino acids into protein in vivo and protein synthesis in vitro in extracts derived from these hybrids is much more sensitive to emetine inhibition than in either emetine-resistant parent. In contrast, no complementation was observed in hybrids derived from fusing two emetine-resistant CHO mutants or in hybrids derived from fusing two emetine-resistant CHL cell lines. These results indicate the CHO emetine-resistant mutants belong to one complementation group and the CHL emetine-resistant mutants belong to another. The genetic loci represented by these two complementation groups must both encode for gene products involved in protein synthesis.

Diphtheria toxin resistance in human lymphoblast lines

Stable mutants (Dip r), highly resistant to diphtheria toxin have been selected from a sensitive human lymphoblast line. A second human lymphoblast line, HH-4 (and its derivative TK6-1) were found to be highly resistant to diphtheria toxin without any previous selection, suggesting the presence of the Dip r allele in the human population. The resistance of protein synthesis in extracts of mutant cells to diphtheria toxin indicates that the genetic lesion in the resistant lines examined involved an alteration in the protein synthesis. In comparison to sensitive cells, the mutant cell extracts contained reduced (30–40%) levels of ADP-ribosylatable elongation factor-2 activity suggesting that the lesion presumably affects elongation factor-2 in such cells. The biochemical phenotype of these mutants appears similar to that of the Dip rIIb class of mutants of Chinese hamster cells (4,6) which behave codominantly in hybrids.

Stimulation of protein synthesis and Met-tRNA binding by phosphorylated sugars: studies of their mechanism of action

It has been previously reported by J. R. Lenz et al. [(1978) Biochemistry 17, 80--87] that certain phosphorylated sugars stimulate protein synthesis in extracts of mammalian cells. This effect was found to be due to a stimulation of Met-tRNAf binding to 40S ribosomal subunits, both in whole extracts and with isolated ribosomes. However, formation of a ternary complex of Met-tRNAf, initiation factor eIF-2, and GTP was not stimulated. It was also shown that the stimulation is not due solely to metabolism of the sugars. The present communication further characterizes the stimulatory effect of the sugars. They were found to prevent the inactivation of ribosomes that occurs during protein synthesis incubations. The sugars were also found to inhibit cAMP-dependent protein kinases noncompetitively. However, they stimulate Met-tRNAf binding to 40S ribosomal subunits even under conditions in which an inhibition of protein kinase has no effect. Although it has bot been possible to demonstrate a direct association of the sugars with the 40S initiation complex, the evidence suggests that their effect is mediated by an interaction with one of the components involved in the formation of this complex.

Synthesis and Breakdown of pppA2′p5′A2′p5′A and Transient Inhibition of Protein Synthesis in Extracts from Interferon‐Treated and Control Cells

Incubation of the mouse L-cell-free system with a concentration of pppA2'p5'A2'p5'A [(2'-5')An] just sufficient to inhibit protein synthesis results in formation of a high-molecular-weight, heatlabile inhibitor and enhanced ribonuclease activity and in the rapid breakdown of (2'-5')An to ATP. The (2'-5')An-enhanced ribonuclease activity is also unstable and in the absence of a (2'-5')-An-regenerating system inhibiton of protein synthesis is transient. Although interferon treatment enhances the synthesis of (2'-5')An, the rates of degradation of (2'-5')An and levels of activatible nuclease are similar in extracts prepared from control or interferon-treated cells. Interestingly, the sensitivity of different cell-free systems to (2'-5')An, varies with the source of the cell-free systems and with the methods used in their preparation. There is, however, no obvious correlation between the sensitivities of the system and the rate of breakdown of (2'-5')An. The significance of these results is discussed in relation to a possible control function for the (2'-5')An system in both interferon-treated and control cells.

Diphtheria-toxin-resistant mutants of CHO cells affected in protein synthesis: a novel phenotype.

Stable mutants highly resistant to the protein-synthesis-inhibitor diphtheria toxin have been selected in Chinese hamster ovary (CHO) cells. Protein synthesis in extracts of mutant cells is resistant to the inhibitory action of diphtheria toxin, indicating that the lesion has affected the protein-synthesis machinery. However, about 50% of the elongation factor-2 (EF-2) activity in the mutant cells can still be ADP-ribosylated by diphtheria toxin, and this remaining EF-2 activity is similar to that present in the wild-type cells. We suggest that this result is best explained by assuming that our CHO cells contain two functional copies of the EF-2 gene, and that only one of the copies is altered in the mutants. According to this view, the mutated allele produces EF-2 resistant to ADP-ribosylation which is capable of supporting cell growth in the presence of diphtheria toxin. Although the Dipr marker seems to act dominantly in the parental CHO cells, its behavior in Dipr X Dips hybrids (CHO X CHO) is recessive as measured by cell survival in presence of the toxin. This paradoxical behavior may be due to a gene dosage effect. Segregation studies from hybrids show that the Dipr marker segregates independently of the Emtr and Thgr markers indicating that the Dipr locus is not linked to either the Emtr locus or to the X chromosome.

Influence of preincubation on the development of the inhibition of protein synthesis in extracts from interferon-treated mouse L cells action on tRNA

Treatment of cells with interferon leads to an inhibition of cell-free protein synthesis in preincubated cell extracts. We show here that the observation of such a defect in the translation of both Mengo virus RNA and poly(U,C) depends critically upon the length of preincubation period. Control and interferon-treated cell extracts (Cont. S-10, Int. S-10) preincubated for up to 60 min have identical protein synthetic activity when assayed with either Mengo virus RNA or poly(U,C). Although preincubation of Cont. S-10 for more than 60 min leads to a gradual reduction in the protein synthetic capacity of the extract, the activity of Int. S-10 declines much more rapidly, and after 60 to 120 min of preincubation the translational activity of Int. S-10 is markedly inhibited (80–90%) compared to Cont. S-10. In extracts preincubated for times sufficient to allow the development of this impaired protein synthesis, addition of both total tRNA and a purified leucine tRNA restores synthesis by Int. S-10 to almost the same level as the corresponding controls. Addition of total tRNA, but not leucine tRNA, also overcomes the loss of activity observed in Cont. S-10 extracts which have been preincubated for more than 60 min. Addition of leucine tRNA to Int. S-10 during the preincubation step strongly reduces the ability of the tRNA to restore protein synthesis. Int. S-10 programmed with Mengo virus RNA synthesized the same polypeptides as Cont. S-10 when the preincubation period was 60 min or less. Although preincubation of Cont. S-10 for more than 60 min led to a decline in their activity, the pattern of polypeptides synthesized was not affected. However, preincubation of Int. S-10 for more than 60 min caused a decrease in the size of polypeptides as well as a reduction in their amount. Only peptides of MW 7000 to 26,000, were detected in such extracts. Addition of tRNA to the Int. S-10 restored the synthesis of larger polypeptides in a dose-dependent fashion and, with suitable amounts of total tRNA, the pattern of polypeptides synthesized was identical to that for control extracts. Our results indicate that preincubation of cell-free extracts leads to a decline in their capacity to utilize tRNA and that pretreatment of the cells with interferon strongly enhances this effect, at least with respect to leucine tRNA. The inability of extracts prepared from interferon-treated cells to utilize endogenous tRNA after prolonged preincubation (60–120 min) is most probably due to inactivation of the tRNA.

Dual Action of Double‐Stranded RNA in Inhibiting Protein Synthesis in Extracts of Interferon‐Treated Mouse L Cells

We have investigated the mechanism by which double‐stranded RNA (dsRNA) inhibits protein synthesis in cell‐free extracts prepared from mouse L cells which had been pre‐treated with purified interferon. Preincubation of such extracts for a determined period of time causes inactivation of tRNA species and hence an inhibition of protein synthesis with either Mengo RNA or poly(U,C) as mRNA. This inhibition can be overcome by addition of tRNA. We show that dsRNA inhibits the translation of Mengo RNA irrespective of the period of preincubation and that this inhibition is not overcome by tRNA. Translation of poly(U,C) is unaffected by dsRNA. Thus dsRNA does not act at the level of peptide chain elongation and does not cause inactivation of tRNA species, indicating that dsRNA inhibits protein synthesis in extracts of interferon‐treated cells by a mechanism which is different from that responsible for the inhibition observed in the absence of dsRNA.The addition of dsRNA to extracts prepared from interferon‐treated cells causes two modifications which are expected to inhibit protein synthesis. (a) An enhancement of the level of nuclease activity, causing Mengo [3H]RNA to be degraded more rapidly in interferon‐treated cell extracts than control extracts, even when association of Mengo RNA with ribosomes is inhibited by the addition of pactamycin. (b) An impairment in the formation of methionyl‐tRNA · 40‐S‐ribosomal‐subunit complexes. The reduced binding of methionyl‐tRNA to 40‐S subunits can be overcome by the addition of a crude initiation factor preparation suggesting that dsRNA causes inactivation of an endogenous initiation factor(s).In the early stages of translation of Mengo RNA (10–15 min) dsRNA causes a reduction in the incorporation of [35S]methionine into the N‐terminal position of polypeptides suggesting that initiation of protein synthesis is impaired, presumably as a result of the block of methionyl‐tRNA · 40‐S‐subunit complex formation. At a later stage (30–45 min) Mengo RNA is almost completely destroyed under the normal conditions of protein synthesis.