Evidence for the absence of the terminal adenine nucleotide at the amino acid-acceptor end of transfer ribonucleic acid in non-lactating bovine mammary gland and its inhibitory effect on the aminoacylation of rat liver transfer ribonucleic acid

1. tRNA isolated from non-lactating bovine mammary gland competitively inhibits the formation of aminoacyl-tRNA in the rat liver system. 2. Non-lactating bovine mammary gland tRNA and twice-pyrophosphorolysed rat liver tRNA are unable to accept amino acids in a reaction catalysed by aminoacyl-tRNA synthetases from either rat liver or bovine mammary gland. Deacylated rat liver tRNA can however be aminoacylated in the presence of either enzyme. 3. Bovine mammary gland tRNA lacks the terminal adenine nucleotide at the 3′-terminus amino acid acceptor end, which can be replaced by incubation in the presence of rat liver nucleotide-incorporating enzyme, ATP and CTP. 4. The enzymically modified bovine tRNA (tRNApCpCpA) can bind labelled amino acids to form aminoacyl-tRNA, which can then transfer its labelled amino acids to growing polypeptide chains on ribosomes. 5. Molecules of rat liver tRNA or bovine mammary gland tRNA that lack the terminal adenine nucleotide or the terminal cytosine and adenine nucleotides inhibit the aminoacylation of normal rat liver tRNA to varying degrees. tRNA molecules lacking the terminal −pCpCpA nucleotide sequence exhibit the major inhibitory effect. 6. The enzyme fraction from bovine mammary gland corresponding to that containing the nucleotide-incorporating enzyme in rat liver is unable to catalyse the incorporation of cytosine and adenine nucleotides in pyrophosphorolysed rat liver tRNA and deacylated bovine tRNA. This fraction also markedly inhibits the action of the rat liver nucleotide-incorporating enzyme.     

Competing addition and hydrolysis of the cytidylylcytidylyladenosine terminal residues of transfer ribonucleic acid isolated from the non-lactating bovine mammary gland

1. The enzyme fraction obtained from the pH5 enzyme of non-lactating bovine mammary gland between 40 and 100% ammonium sulphate saturation markedly inhibited the AMP-incorporating activity of rat liver nucleotide-incorporating enzyme. This inhibitory effect has been attributed to high nuclease activity which can be partially removed by adsorption of the enzyme fraction on to calcium phosphate gel. 2. The degradation action of the calcium phosphate-purified enzyme is confined mainly to the terminal trinucleotide sequence -pCpCpA of tRNA, its effect being analogous to that of venom phosphodiesterase. This enzyme is heat labile and very readily loses its degradative activity. 3. Treatment of the enzyme fraction with Macaloid results in complete removal of the phosphodiesterase, leaving an enzyme capable of incorporating AMP into tRNA. 4. Transfer RNA extracted from non-lactating bovine mammary gland in the presence of polyvinyl sulphate and Macaloid is able to accept amino acids with an efficiency 30% of that shown by lactating bovine mammary-gland tRNA isolated under identical conditions.     

Some biochemical effects of triamcinolone acetonide on rat liver and muscle

1. The powerful anti-inflammatory glucocorticoid triamcinolone acetonide, administered to rats at 20 and 2.5mg/kg, leads to a decrease in the incorporation in vivo of [(3)H]uridine and [(32)P]orthophosphate into hind-limb skeletal muscle. 2. At the higher dose, this decrease in the rate of incorporation of precursors into RNA precedes a decrease in the incorporating ability of muscle ribosomes, which commences about 4-5h after drug administration, but is unaccompanied by any changes in the concentration of tissue ATP or free amino acids. 3. The ribosomal dysfunction extends to polyribosomes, which can only be successfully isolated from the muscle of triamcinolone-treated animals after the addition of alpha-amylase to the tissue homogenate to remove glycogen. 4. The specific radioactivity of muscle protein labelled in vivo with (14)C-labelled amino acids does not decrease progressively after triamcinolone administration. After 2h there is an apparent stimulation of incorporation which leads to an overall discrepancy between measurements of protein-synthetic activity made in vivo and in vitro. 5. There is a significant increase in muscle-glycogen concentration between 8 and 12h after the administration of triamcinolone acetonide (20mg/kg), although a significant decrease occurs after 4h. The fall in glycogen concentration may be due to a decrease in the rate of synthesis of protein essential for glucose uptake into the tissues. 6. As judged by (a) incorporation of (14)C-labelled amino acids into protein, (b) [(3)H]uridine and [(32)P]-orthophosphate incorporation into RNA, (c) the rate of induction of tryptophan pyrrolase and (d) changes in the pool sizes of taurine and tryptophan, the responses in liver followed the same time-course as those in muscle after administration of the drug.     

Protein synthesis by perfused hearts from normal and insulin-deficient rats. Effect of insulin in the presence of glucose and after depletion of glucose, glucose 6-phosphate and glycogen

In the absence of glucose, insulin stimulated the incorporation of (14)C-labelled amino acids into protein by perfused rat hearts that had been previously substantially depleted of endogenous glucose, glucose 6-phosphate and glycogen by substrate-free perfusion. This stimulation was also demonstrated in hearts perfused with buffer containing 2-deoxy-d-glucose, an inhibitor of glucose utilization. It is concluded that insulin exerts an effect on protein synthesis independent of its action on glucose metabolism. Streptozotocin-induced diabetes was found to have no effect either on (14)C-labelled amino acid incorporation by the perfused heart or on the polyribosome profile and amino acid-incorporating activity of polyribosomes prepared from the non-perfused hearts of these insulin-deficient rats, which show marked abnormalities in glucose metabolism. Protein synthesis was not diminished in the perfused hearts from rats treated with anti-insulin antiserum. The significance of these findings is discussed in relation to the reported effects of insulin deficiency on protein synthesis in skeletal muscle.     

Myocardial aminoacyl-transfer-ribonucleic acid synthetase and aminoacyl-transferring enzyme activity

The properties of cytoplasmic aminoacyl-tRNA synthetase and aminoacyl-transferring enzymes in the myocardium were examined and methods for the assay of the activity of these enzyme systems were developed. Aminoacyl-tRNA synthetase activity was measured from the rate of incorporation of ¹⁴C-labelled amino acid into aminoacyl-tRNA. Transferase activity was measured from the rate of incorporation of amino[¹⁴C]acyl-tRNA into protein in the presence of a standard preparation of hepatic ribosomes. Aminoacyl-tRNA synthetase activity is labile once the heart has been homogenized, whereas transferase activity is stable. The source of energy for synthetase activity is ATP; that for transferase is GTP. Transferase activity was inhibited by puromycin and stimulated by dithiothreitol, whereas synthetase activity was unaffected.     

The rate-limiting step of protein synthesis in vivo and in vitro and the distribution of growing peptides between the puromycinlabile and puromycin-non-labile sites on polyribosomes

1. At 3 min after an intravenous injection of radioactive amino acids into the rat, the bulk of radioactivity associated with liver polyribosomes can be interpreted as growing peptides. 2. In an attempt to identify the rate-limiting step of protein synthesis in vivo and in vitro, use was made of the action of puromycin at 0 degrees C, in releasing growing peptides only from the donor site, to study the distribution of growing peptides between the donor and acceptor sites. 3. Evidence is presented that all growing peptides in a population of liver polyribosomes labelled in vivo are similarly distributed between the donor and acceptor sites, and that the proportion released by puromycin is not an artifact of methodology. 4. The proportion released by puromycin is about 50% for both liver and muscle polyribosomes labelled in vivo, suggesting that neither the availability nor binding of aminoacyl-tRNA nor peptide bond synthesis nor translocation can limit the rate of protein synthesis in vivo. Attempts to alter this by starvation, hypophysectomy, growth hormone, alloxan, insulin and partial hepatectomy were unsuccessful. 5. Growing peptides on liver polyribosomes labelled in a cell-free system in vitro or by incubating hemidiaphragms in vitro were largely in the donor site, suggesting that either the availability or binding of aminoacyl-tRNA, or peptide bond synthesis, must be rate limiting in vitro and that the rate-limiting step differs from that in vivo. 6. Neither in vivo nor in the hemidiaphragm system in vitro was a correlation found between the proportion of growing peptides in the donor site and changes in the rate of incorporation of radioactivity into protein. This could indicate that the intracellular concentration of amino acids or aminoacyl-tRNA limits the rate of protein synthesis and that the increased incorporation results from a rise to a higher but still suboptimum concentration.     

Sites of synthesis of plasma proteins in the foetal rat

1. The foetal rat of 16 or more days incorporates ¹⁴C-labelled amino acids into all the demonstrable plasma protein fractions in vivo. 2. Slices of foetal rat liver incubated in vitro incorporate ¹⁴C-labelled amino acids into the main plasma protein fractions, including the foetal-specific `post-albumin'. 3. Slices of placenta are unable to incorporate ¹⁴C-labelled amino acids into plasma proteins in vitro. 4. Liver slices from maternal rats incubated in vitro incorporate ¹⁴C-labelled amino acids into plasma proteins. The presence of post-albumin cannot be demonstrated after incubation. 5. Liver slices from foetal rats, but not from adult rats, contain demonstrable amounts of haemoglobin into which ¹⁴C-labelled amino acids are incorporated.     

PROTEIN SYNTHESIS BY HIGHLY AGGREGATED AND PURIFIED POLYSOMES FROM YOUNG AND ADULT RAT BRAIN

The state of aggregation and the activity of polyribosomes as well as the activity of the pH 5 enzyme fraction were studied at two stages of postnatal brain development, 9 and 50 days after birth. When the polyribosomes were prepared at 0°C in the presence of 5 mm -Mg²⁺, more than 85 per cent of the polyribosome material exhibited a sedimentation coefficient higher than 110 S. High Mg²⁺ concentrations are, therefore, unnecessary to obtain highly aggregated brain polyribosomes. The basal amino acid incorporating activity of both 9- and 50-day-old rat brain preparations is at least equal to that of rat liver. When prepared by the same procedure as above, 9-day-old rat brain polyribosomes seem to be more active (20 per cent) than those of adult brain. However, this difference in activity depends on the presence of a non-ribosomal inactive contaminant which is always present in higher amounts in adult brain preparations. When purified from this contaminant, the preparations do not differ in activity. High Mg²⁺ concentrations are also not necessary for optimal protein synthetic activity and, in fact, are inhibitory. When assayed with both types of highly aggregated polyribosomes, the pH 5 enzyme fraction from adult brain is clearly less active than that of 9-day-old rats. These results suggest that the loss of brain protein synthesis during development does not depend on the stability of the messenger RNA-ribosome complex but only on the soluble pH 5 enzyme fraction.     

Evidence for defective transfer ribonucleic acid in polymyopathic hamsters and its inhibitory effect on protein synthesis

1. Different reaction steps involved in protein synthesis were studied in skeletal muscles from control and myopathic hamsters. 2. There was no difference between partially purified aminoacyl-tRNA synthetases from myopathic and control animals in yield or catalytic activity, as tested with exogenous deacylated tRNA. 3. However, isolated deacylated tRNA from myopathic muscle was aminoacylated by these synthetases to a lesser extent than that derived from control muscle. 4. Addition of deacylated tRNA isolated from control muscle improved the performance of pH5 enzymes from myopathic muscle in polypeptide synthesis on homologous polyribosomes; tRNA isolated from myopathic animals did not. 5. Preparation of extracts from both types of animals in the presence of the ribonuclease-absorbent bentonite led to an increased capacity of endogenous tRNA to accept amino acids in pH5 enzymes prepared from normal and abnormal tissue, but the difference between the two systems remained the same. 6. Total tRNA nucleotidyltransferase activity, tested with twice-pyrophosphorolysed rat liver tRNA, was identical in both extracts. 7. Added tRNA nucleotidyltransferase incorporated more AMP and CMP into endogenous tRNA with the pH5 enzyme from myopathic muscle than with that from control muscle. 8. Preincubation of deacylated tRNA from myopathic muscle with ATP, CTP and tRNA nucleotidyltransferase more than doubled its subsequent aminoacyl-acceptor activity, and halved the extent of the defect relative to aminoacylation of control tRNA similarly treated. Endogenous tRNA in pH5 enzyme preparations behaved likewise. 9. It is suggested that a 3'-exonuclease in myopathic muscles attacks tRNA molecules in such a way that some of them remain substrates for tRNA nucleotidyltransferase, which may incorporate into RNA not only AMP and CMP, but also GMP. 10. Cell-free protein synthesis in preparations from myopathic hamster muscles is limited by the supply of intact tRNA molecules.     

Effect of aromatic acids on protein synthesis in subcellular preparations from the rat brain.

The incorporation of [3H]phenylalanine, [3H]tyrosine, and [3H]tryptophan into protein and amino acyl-tRNA was studied in cell-free preparations from rat brain. Tyrosine and tryptophan inhibited the incorporation of phenylalanine into protein, and tyrosine inhibited the incorporation of phenylalanine and tryptophan into amino acyl-tRNAs. In most cases, homogentisate, phenylpyruvate, and phenyllactate inhibited the incorporation of phenylalanine, tyrosine, and tryptophan into protein and amino acyl-tRNAs, and the incorporation of phenylalanine into polyphenylalanine. All other protein amino acids, and phenylacetate, salicylate, and benzoate were wholly ineffectual. The results suggest that the formation of amino acyl-tRNAs may have been the step which was affected most by the inhibitors. The incorporation data at different concentrations of the aromatic amino acids were fitted to the simple Michaelis equation. Homogentisate and phenylpyruvate generally tended to reduce both Km and V in the incorporation of aromatic amino acids into protein and amino acyl-tRNAs, even if V decreased more than Km.     

Effect of thyroidectomy on pathways of glucose metabolism in lactating rat mammary gland

1. Assessment of the overall metabolic changes in lactating mammary gland after thyroidectomy has been made by measurement of the incorporation of (14)C from specifically labelled glucose, pyruvate and acetate into (14)CO(2) and (14)C-labelled lipid in the experimental rats and in sham-operated control animals. 2. Thyroidectomy depressed the oxidation of (14)C-labelled substrates, an effect still apparent when the control rats were pair-fed with thyroidectomized rats; however, the ratio of oxidation of [1-(14)C]glucose/oxidation of [6-(14)C]glucose was unaltered. In parallel with these studies it was revealed that the activities of hexokinase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADP-linked isocitrate dehydrogenase were all lower in the thyroidectomized group than in the pair-fed control group. 3. Thyroidectomy also lowered the incorporation of (14)C-labelled substrates into (14)C-labelled lipid, an effect further studied by measurement of the activities of citrate-cleavage enzyme and acetate thiokinase. Restricting the food intake of the control rats to that of the thyroidectomized group lowered the activity of citrate-cleavage enzyme, but no further depression was observed on thyroidectomy. The oxidized and reduced nicotinamide nucleotide content of mammary tissue was shown to be decreased in the thyroidectomized rats compared with the control rats.     

The binding of radioactive label from labelled phenacetin and related compounds to rat tissues in vivo and to nucleic acids and bovine plasma albumin in vitro

1. acetyl-(3)H- and ethyl-(14)C-labelled derivatives of phenacetin and related compounds are described. 2. Radioactive label from the ethyl-(14)C-labelled derivatives of 4-nitrophenetole, 4-phenetidine and phenacetin binds in vitro to various extents to bovine plasma albumin, salmon sperm DNA and yeast RNA; the extent of binding is increased in the presence of a rat liver microsomal hydroxylating system and further increased when the microsomal enymes are induced by prior treatment of rats with 3-methylcholanthrene. 3. The ratios of the bound radioactive labels in vitro from [ethyl-(14)C]phenacetin, N-acetoxy[ethyl-(14)C]phenacetin, [acetyl-(3)H]phenacetin and [diacetyl-(3)H]N-acetoxyphenacetin per g-atom of DNA P, RNA P and per mol of protein in the absence of the microsomal system are approximately 1:60:11:863, 1:68:41:1835 and 1:88:713:2399 respectively. 4. Radioactive label from labelled phenacetin binds in vitro to all tissues examined, including the spleen, intestines, kidney and bladder; about 80% of the radioactivity bound to the liver is concentrated in the RNA and proteins. 5. Comparison of the relative extents of binding of radioactive label derived from equimolar amounts of labelled phenacetin, ethanol or acetate shows that the incorporation of labelled C(2) units into tissues and biological macromolecules in vivo and in vitro may account for only a part of the total bound radioactive label derived from phenacetin and not at all from the incorporation of radioactive acetate into nucleic acids. 6. Some implications of these findings are discussed.     

Evoked release from guinea pig cerebral cortex slices of endogenous 14C-labelled amino acids, labelled via D-[U-14C]glucose.

Release of endogenous amino acids labelled via D-[U-14C]glucose was compared with that of several exogenous labelled amino acids using slices of guinea pig cerebral cortex. Electrical field stimulation evoked a selective release of endogenous [14C]glutamate, [14C]aspartate, and gamma-amino[14C]butyrate (14C-labelled GABA). The selectivity of release correlated well with 14C incorporation into endogenous amino acids. Calculations of the fraction of the tissue radioactivity released indicated that the selectivity was not an artifact due to differential incorporation. Because glucose in mammalian brain is metabolized almost entirely by the so-called 'large compartment', it is tentatively concluded that the releasable 'transmitter pool' of glutamate, aspartate, and GABA is located in this 'large compartment'.     

The site at which 4-iodoacetamidosalicylate reacts with glutamate dehydrogenases

1. Bovine, porcine and chicken liver glutamate dehydrogenases were irreversibly inhibited by a tenfold excess of radioactive 4-iodoacetamidosalicylic acid at pH7.5. 2. Inhibition was accompanied by the covalent incorporation of 1.1 mol of labelled inhibitor/mol of polypeptide chain. Acid hydrolysis yielded N^ε-carboxymethyl-lysine as the sole labelled amino acid. No labelled S-carboxymethylcysteine was recovered from the bovine or porcine enzymes. 3. The labelled bovine enzyme was hydrolysed with trypsin. The radioactivity was found at lysine-126 in a peptide comprising residues 119–130 of the sequence. 4. The amino acid compositions of the tryptic peptides containing labelled lysine from the porcine and chicken enzymes were similar to that of the bovine peptide.     

Partial purification and properties of mammalian phosphatidylglycerophosphatase

The phosphatidylglycerophosphatase (EC 3.1.3.27) activity of rat liver mitochondria was investigated by assaying the conversion of 14C-labelled phosphatidylglycerophosphate to phosphatidylglycerol. The activity was associated with a mitochondrial membrane fraction and could not be released into solution employing techniques applicable to a peripheral membrane protein. The enzyme was partially purified by sonication, pH 5.0 precipitation, and gel filtration. Various ionic and nonionic detergents as well as numerous divalent cations inhibited the phosphatase. The enzyme displayed a high affinity for phosphatidylglycerophosphate.     

Polyribosomes, isolated from rat liver in a low ionic strength medium, capable of autonomic translation in a cell-free system without the addition of cellular fluid

Polyribosomes isolated from the liver in the presence of 10 mM KCl and purified by centrifugation through 2 M sucrose were shown to incorporate [3H]leucine both into aminoacyl-tRNA and polypeptides in a cell-free system without cell sap. The incorporation of [3H]leucine showed a linear increase within 80-100 min and was then levelled off. The system was sensitive to cycloheximide, puromycin and ethionine and needed ATP, GTP and unlabeled amino acids. The quantitation of tRNA in polyribosomes (the fraction which did not sediment with the subparticles after polyribosome dissociation) revealed more than two tRNA molecules per 80S monosome. It is likely that this tRNA excess as well as the earlier established presence of aminoacyl-tRNA synthetases and elongation factors promote the autonomic translation of polyribosomes.     

The effect of 4-dimethylamino-3′-methylazobenzene on 14C-labelled amino acid incorporation by rat-liver polysome preparations

1. The incorporation of (14)C-labelled amino acids into polysomal protein was studied in a system consisting of polysomes and pH5 enzyme obtained 4 and 40hr. after a single intraperitoneal injection of 4-dimethylamino-3'-methylazobenzene. Labelling of the polysome fraction of preparations of both the 4hr.-treated and 40hr.-treated rats was considerably higher than in the normal control. 2. In further experiments on protein synthesis by polysomes from azo-dye-treated rats, the effect of replacing pH5 enzyme with cell sap was studied. Incorporation of (14)C-labelled amino acids into polysomal protein was similar to that of the control. 3. Aggregate size of polysomes obtained from rats injected previously with 4-dimethylamino-3'-methylazobenzene was studied by sucrose-gradient centrifugation. Polysomes prepared at 4hr. after azo-dye administration contained a considerable amount of large aggregates (approx. 700s), whereas at 40hr. after administration of the azo-dye the amount of large aggregates was less than in the control. 4. Determination of the ultraviolet spectra of polysome preparations from both normal and azo-dye-treated rats revealed no difference between the preparations. On the other hand, the ultraviolet spectra of cell-sap fractions from the different preparations showed that there is a definite shift in the absorption maximum from 272mmu (normal) to 267mmu, 40hr. after treatment, with an intermediate value of 270mmu for the preparation from 4hr.-treated rats. The absorption minimum changes from 250mmu (normal) to 245mmu for the preparation from 40hr.-treated rats.     

Protein synthesis in tomato-fruit locule tissue: Incorporation of amino acids into protein by aseptic cell-free systems

1. Osmotically disrupted protoplasts and isolated plastids from tomato-fruit locule tissue were found capable of incorporating (14)C-labelled amino acids under aseptic conditions into an exhaustively washed trichloroacetic acid-insoluble protein fraction. 2. The disrupted protoplast system incorporated 20-45mumumoles of amino acid/mg. of protein in 10min. The isolated plastid system incorporated 10-20mumumoles of amino acid/mg. of protein; 40-150mumug. of carbon/mg. of protein was incorporated in 10min. from (14)C-labelled amino acid mixture. 3. Incorporation is stimulated by added ATP in the dark, but no added ATP is required when the system is illuminated. The cell-free plastid system is to some extent self-sufficient and does not normally require an added supernatant fraction or unlabelled amino acids. 4. Amino acid incorporation by plastids is inhibited by chloramphenicol, puromycin, actinomycin D, ribonuclease and deoxyribonuclease. It is suggested that the mechanism of protein synthesis in the cell-free plastids, and in the tissue generally, is basically the same as established for bacteria. Ribosomes and highspeed supernatant from this tissue were to some extent interchangeable with Escherichia coli ribosomes and supernatant in cell-free incubations. 5. Incorporation of amino acids by isolated plastids was stimulated by indol-3-ylacetic acid and kinetin, and, whereas incorporation normally proceeds for only 10-20min., the time-course was extended in the presence of these growth substances. It is suggested that hormones may be involved in the regulation of protein synthesis in plants.     

Biosynthesis in vitro of tryptophanase by polyribosomes from induced cultures of Escherichia coli

1. Polyribosomes were isolated from Escherichia coli grown in media in which tryptophanase is induced and in which it is repressed. The polyribosomes from the induced bacteria had a small amount of tryptophanase activity associated with them. 2. A portion of the enzyme activity remained bound to polyribosomes during centrifuging in sucrose gradients. 3. Incubation of tryptophanase-containing polyribosomes with puromycin released enzyme activity. 4. The binding of the enzyme to the polyribosomes did not depend on the presence of DNA. 5. When the polyribosomes were incubated under conditions of protein synthesis with supernatant fraction obtained from repressed bacteria, a small but statistically significant increase in enzyme activity was produced. 6. When a radioactive amino acid was included in the incubation mixture for the tryptophanase system a radioactive protein was obtained whose chromatographic, electrophoretic and sedimentation properties were identical with those of tryptophanase. 7. The amount of incorporation was consistent with the amount of new enzyme synthesis predicted by the increase in enzyme activity. Both radioactive incorporation and increase in enzyme activity were shown to be energy-dependent and also negative controls were obtained by using zero-time incubations or polyribosomes isolated from either repressed cells or a mutant lacking the ability to produce tryptophanase. 8. The distribution of radioactive leucine in the carboxyl region of the newly labelled tryptophanase was examined by digesting the labelled protein with carboxypeptidases. It was shown that the radioactivity was more highly concentrated towards the carboxyl terminus when the incubation times for protein synthesis were shorter (implying that, with longer incubation times, longer lengths of polypeptide chain contained radioactive amino acid residues).     

Effect of some polycyclic aromatic hydrocarbons on protein synthesis in vitro

1. The effect of the strongly carcinogenic polycyclic aromatic hydrocarbons benzo[a]pyrene, 3-methylcholanthrene and dibenz[a,h]anthracene and of the non-carcinogenic anthracene, pyrene and phenanthrene on protein synthesis was studied in vitro with subcellular systems from rat liver. 2. Both types of hydrocarbons affect amino acid activation and inhibit transfer of labelled amino acids from transfer RNA to ribosomes. 3. Only the carcinogenic compounds stimulate the incorporation of labelled algal-protein hydrolysate and of some individual amino acids into transfer RNA. The most active dose was 10mmug. under the conditions used. This effect is abolished by preincubation of pH5 enzymes with the carcinogens before the addition of radioactive amino acids. 4. The carcinogens stimulate the incorporation of some amino acids into ribosomal protein whereas the non-carcinogenic compounds have no such effects. 5. Polynucleotide-dependent stimulation of protein synthesis is greatly enhanced in the presence of the carcinogenic hydrocarbons when either free amino acids or transfer RNA charged with labelled amino acids are used. The non-carcinogenic compounds induce a partial inhibition of this process. 6. It is concluded, in agreement with other authors, that carcinogens may increase the number of active incorporation sites on both transfer and ribosomal RNA. Possible mechanisms of such an effect are discussed.