Stimulation of globin-chain initiation by hemin in the reticulocyte cell-free system.

109

The control of globin synthesis by hemin in cell-free preparations of rabbit reticulocytes is mediated by an inhibitor (translational repressor) of globin chain initiation that is inactivated by hemin. When the ribosome-free supernatant fraction is warmed at 34°with-out hemin, it quickly acquires the ability to inhibit a fresh cell-free preparation. If the warmed supernatant fraction is further incubated with hemin, its inhibitory activity is lost. This reversible inhibitor, which is observable only during the early period of incubation without hemin, is distinct from an irreversible inhibitor that becomes prevalent during longer incubations. The reversible inhibitor may be an intermediate in the formation of the irreversible inhibitor. The sensitivity of the reversible inhibitor to inactivation by hemin, which permits resumption of globin synthesis in both intact cells and lysates, indicates that the inhibitor is a physiological regulator.

mentioning

confidence: 99%

Control of Globin Synthesis in Cell-Free Preparations of Reticulocytes by Formation of a Translational Repressor That is Inactivated by Hemin

Gross

Rabinovitz

1972

109

“…Rabbit reticulocytes are particularly suitable to answer these questions, because in a cellfree system prepared from them, ribosomes can recycle more than fifty times over endogenous messenger RNA (7); furthermore, when this system is incubated at 280 or higher, initiation of protein synthesis (mostly globin) decreases drastically after 5 min, and polysomes disappear, unless heme is added (7)(8)(9)(10)(11).…”

mentioning

confidence: 99%

Translational Control of Hemoglobin Synthesis by an Initiation Factor Required for Recycling of Ribosomes and for their Binding to Messenger RNA

Kaempfer

Kaufman

1972

The continued recycling of ribosomes during protein synthesis in rabbit reticulocyte lysates at 370 requires an initiation factor whose activity is rapidly lost in the absence of added heme. Partially purified factor (i) fully maintains the polysomes; (ii) inhibits the association of 40S and 60S ribosomal subunits into single ribosomes; (iii) promotes the quantitative entry of added 60S subunits into polysomes; (iv) allows the accumulation of ribosomal subunits, instead of single ribosomes, when initiation is blocked with aurin tricarboxylate; and (v) is absolutely required for the binding of globin messenger RNA to ribosomes.These properties suggest that this mammalian initiation factor functions analogously to bacterial IF-3. In addition, the translational control of globin synthesis by heme is exerted, directly or indirectly, through this factor.Ribosomes in growing bacteria and in the cytoplasm of yeast frequently undergo exchange of their large and small ribosomal subunits (1, 2), apparently by dissociation after each passage over messenger RNA and reformation from a pool of ribosomal subunits that continuously recycle through polysomes (3,4). When protein synthesis slows down, synthetically inactive single ribosomes (lacking mRNA and growing polypeptide chains) accumulate at the expense of polysomes in both prokaryotic and eukaryotic cells. Bacterial 30S and 50S ribosomal subunits possess an extremely high affinity for each other and readily associate to form single ribosomes (5); this association is specifically inhibited by initiation factor IF-3 (5, 6). We have proposed that this is the mechanism by which bacterial IF-3 maintains the recycling or ribosomal subunits through polysomes and prevents their accumulation as single ribosomes (5).We have asked if an analogous factor operates in the mammalian ribosome cycle, and, if so, whether it plays any role in exerting translational control. Rabbit reticulocytes are particularly suitable to answer these questions, because in a cellfree system prepared from them, ribosomes can recycle more than fifty times over endogenous messenger RNA (7); furthermore, when this system is incubated at 280 or higher, initiation of protein synthesis (mostly globin) decreases drastically after 5 min, and polysomes disappear, unless heme is added (7-11).Our finding is that rabbit reticulocytes indeed contain a factor activity that regulates the flow of ribosomal subunits through the ribosome cycle in a manner strictly analogous to bacterial IF-3. This factor, tentatively named IF-3rr, is absolutely required for the binding of globin mRNA to ribosomes.Abbreviation: rr, rabbit reticulocyte.In the absence of added heme, reticulocyte ribosomes fail to recycle through polysomes, unless IF-3rr is added. Directly or indirectly, therefore, translational control of reticulocyte protein synthesis by heme must be exerted through this factor. RESULTS Control of reticulocyte ribosome cycle by the factorThe sedimentation distribution of ribosomes in a 32P-labeled rabbit reticulocyte l...

“…Effects of ATA on the initiation of globin synthesis Crude reticulocyte lysates, capable of initiating new globin chains in the presence of hemin (11), were used to study the effects of ATA on protein synthesis in extracts prepared from a mammalian cell. As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Aurintricarboxylic Acid: Inhibitor of Initiation of Protein Synthesis

Stewart

Grollman

Huang

1971

112

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...