Inhibition by pactamycin of the initiation of protein synthesis. Binding of N-acetylphenylalanyl transfer ribonucleic acid and polyuridylic acid to ribosomes

Cohen, Linda B.; Herner, Albert E.; Goldberg, Irving H.

doi:10.1021/bi00832a004

Cited by 55 publications

(24 citation statements)

References 19 publications

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“…Peptides formed in the presence of ATA and subsequently released from the polyribosome appear to be complete, as shown in the present study and, previously, by Webster and Zinder (15). Taken together, these observations distinguish the effects of ATA from those of other reported inhibitors of the initiation of protein synthesis such as pactamycin (16), cycloheximide (17,18), and sodium fluoride (17,19).…”

Section: Discussionsupporting

confidence: 87%

Aurintricarboxylic Acid: Inhibitor of Initiation of Protein Synthesis

Stewart

Grollman

Huang

1971

Proc. Natl. Acad. Sci. U.S.A.

112

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Aurintricarboxylic acid prevents the attachment of bacteriophage messenger RNA to ribosomes. As a consequence, initiation of protein synthesis in cellfree extracts prepared from Escherichia coli or rabbit reticulocytes is inhibited at concentrations of dye that do not prevent chain extension. Its properties can be distinguished from other agents that inhibit protein synthesis, including sodium fluoride, cycloheximide, and pactamycin.Aurintricarboxylic acid (ATA) is a triphenylmethane dye that prevents attachment of bacteriophage mRNA to Escherichia coli ribosomes but does not dissociate complexes composed of ribosomes, mRNA, formymethionyl-tRNA, and initiation factors (1). The results reported in the present paper indicate that the primary inhibitory effect of ATA in cell-free extracts prepared from E. coli or rabbit reticulocytes is on the initiation of protein synthesis. Protein synthesis is inhibited at concentrations of dye that show little or no effect on the rate or extent of chain extension; the inhibition is accompanied by sequential release of ribosomes and completed peptides from the polyribosomes. MATERIALS AND METHODSThe preparation and sources of ATA, bacteriophage f2 RNA, and the other materials used in these experiments are described elsewhere (1-3). As prepared, ATA is impure and contains significant quantities of isomeric and inactive materials.[14C ]hemoglobin, 4 A&Ci/mg, was prepared by incubating intact rabbit reticulocytes for 24 hr at 30'C in medium (3) containing [14C ]leucine. Preincubated, dialyzed S-30 extracts (1), containing 30-35 mg of ribosomes per ml, were centrifuged at 150,000 X g for 2 hr and the top half of the supernatant solution (S-150) removed. Reticulocyte lysates were prepared and the synthesis of globin was measured, as described by Maxwell and Rabinovitz (4). Density gradient centrifugation (5), acrylamide gel electrophoresis (6), and the determination of radioactivity in precipitates collected on Millipore membrane filters (3) or on crushed acrylamide gels (6) were performed according to published procedures. RESULTSEffects of ATA on initiation of protein synthesis in extracts of E. coli:The inhibitory eftects of ATA on f2 RNA-directed peptide synthesis in extracts of E. coli are shown in Fig. 1A. If the dye is added prior to the addition of f2 RNA, complete inhibition of peptide synthesis is observed. On the other hand, if the same concentration of ATA is added 10 or 15 min after initiating the reaction, the rate of protein synthesis remains unaffected for several minutes, then decreases rapidly. In contrast to this delayed inhibitory effect of ATA, chloramphenicol, an antibiotic that inhibits chain elongation (7), stops peptide synthesis immediately after addition to the reaction mixture (Fig. 1B).Effects of ATA on the synthesis of phage coat and other proteins The effects of ATA on the synthesis of coat and non-coat proteins are compared in the experiments shown in Fig. 2. Synthesis of coat protein is measured by the incorporation of valine (8); synthesis of...

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Section: Discussionsupporting

confidence: 87%

Aurintricarboxylic Acid: Inhibitor of Initiation of Protein Synthesis

Stewart

Grollman

Huang

1971

Proc. Natl. Acad. Sci. U.S.A.

112

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“…Effect of pactamycin on protein synthesis and polysome size distribution in HTC cells Inhibition of protein synthesis in HTC cells by pactamycin was strongly concentration dependent between 0.1 and 1 MAM, as has been reported in other systems (2,13). A concentration of 2 ,M was chosen for the experiments reported below.…”

Section: Resultsmentioning

confidence: 94%

“…This conclusion depends on the demonstration that in HTC cells, as in other cells (2,13), pactamycin preferentially inhibits polypeptide-chain initiation over elongation. This is evident for total protein synthesis from direct NHrterminal analysis (Table 1) as well as from the rate of amino-acid incorporation in the presence of the antibiotic (Fig.…”

Section: Discussionmentioning

confidence: 99%

Mechanism of Hormonal Induction of Tyrosine Aminotransferase Studied by Measurement of the Concentration of Growing Enzyme Molecules

Scott

Shields

Tomkins

1972

Proc. Natl. Acad. Sci. U.S.A.

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Proteins labeled in cultured hepatoma cells grown in the presence of radioactive amino acids and pactamycin consist primarily of growing polypeptide chains initiated before addition of the antibiotic. Nascent tyrosine aminotransferase (EC 2.6.1.5) molecules were labeled in this way, and their radioactivity after completion was measured immunologically. Cells exposed to the synthetic adrenal steroid, dexamethasone, contain 10-to 18-times as much nascent enzyme as uninduced cells. These results indicate that induction control is not at the level of polypeptide-chain elongation or release, but probably operates on either the amount of messenger RNA or the rate of specific polypeptide-chain initiation.Addition of certain adrenal steroids to a line of cultured rat hepatoma (HTC) cells causes an increase in the de novo rate of synthesis of the enzyme, tyrosine aminotransferase (EC 2.6.1.5) (TAT) (1). This increase might be due to (i) an increase in the amount of TAT-specific mRNA, (ii) an increase in the number of ribosomes translating an unchanged amount of TAT mRNA, (iii) an increase in the rate at which each ribosome traverses the TAT mRNA, or (iv) removal of a block in translation behind which ribosomes have accumulated. Possibilities (i) and (ii) imply that more nascent chains of TAT are present in hormonally induced than in uninduced cells; while (iii) and (iv) would mean that nascent TAT chains would remain at the same concentration, or even decrease, when TAT synthesis was induced. To investigate these possibilities and to quantitate the growing chains of TAT, we have taken advantage of the fact that pactamycin prevents initiation but not completion of polypeptide chains (2).Therefore, by addition of radioactive amino acids to a growing HTC-cell culture simultaneously with pactamycin, it is possible to label only chains that had been initiated before the inhibitor was added. The radioactivity incorporated into completed TAT under these conditions gives an estimate of the concentration of nascent enzyme molecules.The experiments reported below show that steroid-induced cells contain an increased concentration of nascent TAT molecules roughly in proportion to the increased rate of enzyme synthesis. From these results, it seems likely that induction is due either to an increased amount of specific message or to an increase in the specific initiation of the messenger for TAT. Cell Growth, Induction of TAT, and Incorporation of Amino Acids. Suspension cultures of HTC cells were grown in modified S-77 medium containing no fetal-calf serum and 10% calf serum (3). Synthesis of TAT was induced with dexamethasone (4). Cells were collected for labeling after 15-20 hr of growth, when they had reached a density of 4-6 X 105/ml. 1-2 hr before the cells were collected, one-tenth volume of fresh growth medium was added, and incubation at 370 was continued. Cells were then harvested, washed in labeling medium (see below), and suspended at a density of 5 X 107/ml. Induced cells were pooled and incubated in labeling medi...

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“…Interestingly, alterations in S2 confer resistance to the antibiotic kasugamycin (16,27), an inhibitor of translation initiation (15,23). Furthermore, photoaffinity labeling studies show that S2 associates with the antibiotic pactamycin (24), which also inhibits translation initiation (2,23). If kasugamycin and pactamycin inactivate S2 or ribosomes containing S2, cI translation may be resistant to inhibition by these antibiotics.…”

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confidence: 99%