Aurintricarboxylic Acid: Inhibitor of Initiation of Protein Synthesis

Abstract

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...

Reconstitution of functional mRNA-protein complexes in a rabbit reticulocyte cell-free translation system.

“…5,1985 on Figure 5A shows that protein synthesis was completely inhibited by aurintricarboxylic acid and edeine, whereas a control reaction showed the normal amount of protein synthesis. Aurintricarboxylic acid inhibits mRNA binding to ribosomes (38), and we anticipated that much of the mRNP formed under these conditions would sediment at less than 40S. Therefore, we recovered and analyzed the <40S material as well as the more rapidly sedimenting material (Fig.…”

Interaction of Escherichia coli 30S Ribosomal Subunits with MS2 Phage RNA in the Absence of Initiation Factors

“…As shown in Table 3, the antibiotic edeine, which inhibits ribosomal binding of fMet-tRNA (21), had no effect on the mRNA binding described here. On the other hand, aurintricarboxylic acid, known to inhibit the binding of mRNA to ribosomes (22) Fig. 2.…”

Anionic Compounds as Inhibitors of African Swine Fever Virus Replication in Vero Cells

“…Several modes of action have been reported for ATA.lt is reported to inhibit the attachment of messenger ribonucleic acid to ribosomes (Grollman and Stewart, 1968) thus preventing initiation of protein synthesis (Blumenthal and Landers, 1973;Huang and Grollman, 1972;Stewart et al, 1971;Siegelman and Apirion, 1971).ATA has also been reported. to inhibit RNA-directed DNA polymerase associated with Raucher murine leukaemia virus (Liao et al, 1975) and avian myeloblastosis virus (Leis and Hurwitz, 1972) and to interfere with the ability of Moloney leukaemia RNA-directed DNA polymerase to bind a DNA primer molecule (Givens and Manly, 1976).…”