Inhibition Of Polypeptide Chain Initiation Research Articles

Regulation of protein synthesis by hemin Effect of dithiothreitol on the formation and activity of the hemin-controlled translational repressor

Previous studies have demonstrated that the hemin-controlled translational repressor (HCR), a high molecular weight protein inhibitor of polypeptide chain initiation in rabbit reticulocyte lysate, is formed from a presynthesized prorepressor over a period of 12–18 h in three stages denoted reversible, intermediate, and irreversible. The prorepressor can, however, be completely converted to irreversible HCR within 2 min by incubation with such sulfhydryl reagents as N-ethylmaleimide. The results in this report demonstrate that dithiothreitol, which stabilizes thiol groups, will, like hemin, prevent the conversion of the prorepressor to HCR and will inactivate reversible HCR. Unlike hemin, dithiothreitol also inactives the intermediate form of HCR. Neither dithiothreitol nor hemin has any effect on the activity of irreversible HCR. Since the prorepressor used in these experiments had been separated from the supernatant factor (a soluble protein that reverses the inhibition of protein synthesis due to HCR), the effect of dithiothreitol and of hemin is independent of this factor and may be mediated by direct interaction with the prorepressor and HCR. Dithioerythritol, the erythro isomer of dithiothreitol, is as effective as dithiothreitol in preventing the formation of HCR, whereas glutathione and β-mercaptoethanol have little or no effect.

Translational control by protein kinase in Artemia salina and wheat germ.

The catalytic subunit of cyclic 3':5'-AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) inhibits translation in Artemia salina and wheat germ extracts. It acts, as in reticulocyte lysates [Datta, A., de Haro, C., Sierra, J. M. & Ochoa, S. (1977) Proc. Natl. Acad. Sci. USA 74, 1463-1467] by catalyzing the conversion of a proinhibitor to an inhibitor of polypeptide chain initiation. Addition of ATP and either cyclic AMP or catalytic subunit promotes the proinhibitor-inhibitor conversion in crude proinhibitor preparations from A. salina embryos. The effect of cyclic AMP is due to stimulation of cyclic AMP-dependent protein kinase, present in such preparations, and is inhibited by hemin. In similar preparations from wheat germ, addition of ATP and catalytic subunit promoted proinhibitor-inhibitor conversion, but addition of ATP and cyclic AMP has little or no effect. As assayed with histone as substrate, wheat germ preparations exhibit a protein kinase activity that is not stimulated by the addition of cyclic AMP or cyclic GMP. Our results suggest that a translational control system, similar to that existing in rabbit reticulocytes and other mammalian cells, is present in organisms evolutionarily far removed from mammals.

The effect of ischaemia and recirculation on protein synthesis in the rat brain.

Abstract— Rats were subjected to cerebral compression ischaemia for 15min and were subsequently recirculated with blood for periods up to 3 h. In vivo incorporation of intravenously administered L‐[1–14C]valine into total brain proteins was found to be severely inhibited (about 20% of controls) after 45 min of recirculation. After 3 h, protein synthesis had increased, the specific radioactivity of proteins then being about 40% of controls. The post‐ischaemic inhibition of protein synthesis was accompanied by a breakdown in polyribosomes and a concomitant increase in ribosomal subunits. In vitro incorporation of L‐[1–14C]phenylalanine by a postmitochondrial supernatant system derived from animals subjected to 15 min ischaemia and 15 min recirculation was also severely reduced and showed, in contrast to control animals, no response to the addition of a specific inhibitor of polypeptide chain initiation (Poly(I)). Together with the in vivo accumulation of ribosomal subunits this indicates a block in peptide chain initiation during the early stages of recirculation.Polyribosomes from animals subjected to 15 min ischaemia without recirculation showed a normal rate of in vitro protein synthesis which was inhibited by Poly(I) to a similar extent as polyribosomes from control animals. These results suggest that the post‐ischaemic inhibition in chain initiation develops during the early stages of recirculation rather than during the ischaemic period itself.

Isolation of two initiation factors that can partially reverse the inhibition of protein synthesis due to hemin deficiency or the hemin-controlled translational repressor in rabbit reticulocyte lysates

The high salt wash of rabbit reticulocyte ribosomes contains two separate factors which can partially reverse the inhibition of polypeptide chain initiation that results when reticulocyte lysate is incubated in the absence of hemin. These two factors, termed initiation factor (IF) 1 and IF-2, have been separated from each other by chromatography on diethylaminoethyl cellulose and then further purified on hydroxyapatite. IF-1 forms a GTP-dependent complex with methionyl-tRNA f that is retained on Millipore filters. When these factors are added to a system containing reconstituted, salt-extracted ribosomes, IF-1 promotes the binding of methionyl-tRNA f to the 40 S subunit, whereas IF-2 promotes the formation of 80 S initiation complexes from 40 S complexes. Addition of small amounts of one factor and a saturating level of the other to the unfractionated lysate and incubation in the absence of hemin produce an additive stimulation of protein synthesis. Each factor can also partially reverse the inhibitory effect of the hemin-controlled translational repressor. The implication of these findings for the mechanism of hemin control of protein synthesis in reticulocyte lysates is discussed.

Characterization fo a macromolecular inhibitor of polypeptide chain initiation from ehrlich ascites tumor cells

Summary A non-dialyzable inhibitor of protein synthesis has been partially purified from Ehrlich ascites tumor cells. Preparations of this inhibitor have no effect on the initial rate of protein synthesis in rabbit reticulocyte lysates but cause a partial shut-off of amino acid incorporation after a lag period. The inhibitor has no appreciable endonuclease activity towards reticulocyte polysomes but under conditions of protein synthesis if causes disaggregation of polysomes. The effect of the inhibition can be overcome by the initiation factor which forms a ternary complex with Met-tRNA f and GTP and which binds of 40S ribosomal subunits. These results suggest that the Ehrlich ascites inhibitor is very similar to an inhibitor previously identified in reticulocytes.

Inhibition of polypeptide chain initiation in Escherichia coli by elongation factor G.

We have previously reported the isolation from E. coli of a specific inhibitor of polypeptide chain initiation that is rendered ineffective when active aminoacylation of transfer RNA is taking place; this is normally the case during natural messenger RNA translation. Surprisingly, the inhibitory activity appears to be a hitherto unrecognized property of the chain elongation factor G. The following hold for preparations purified for either translocase or inhibitor activity: (1) equal electrophoretic mobility on polyacrylamide gels; (2) equal specific activities for (a) inhibition of initiation, (b) translocation, and (c) ribosome-dependent, uncoupled GTPase; and (3) similar heat sensitivity of translocase and inhibitor activities in a temperature-sensitive E. coli mutant with an altered elongation factor G. Different sites are apparently involved in translocation and inhibition because the former, but not the latter, is sensitive to p-chloromercuribenzoate and fusidic acid.

A specific inhibitor of polypeptide-chain initiation in Escherichia coli.

An inhibitor of polypeptide-chain initiation was isolated from E. coli cells. This protein inhibits formation of the 30S or 70S initiation complex with either fMet-tRNA(f) as initiator and AUG, MS2 RNA, or late T4 RNA as messenger, or acPhe-tRNA as initiator and poly(U) as messenger. Chain elongation, e.g., poly(U) translation at high Mg(2+) concentration, is not inhibited. The inhibitor is rendered ineffective when active aminoacylation of tRNA is taking place, e.g., during natural mRNA translation. This inhibitor is distinct from the so-called interference (i) factors, which interfere exclusively with the action of initiation factor 3. Since the new inhibitor can apparently be turned on and off, it may have a regulatory function in translation.

Further studies with labelled polypeptide-chain-initiation factor 3 (IF-3).

The preparation of 35S‐labelled initiation factor 3 (IF‐3) from Escherichia coli is described. As reported earlier, labelled IF‐3 binds to 30‐S, but not to 50‐S ribosomal subunits or 70‐S ribosomes, and is released upon formation of a 70‐S couple. In continuation of this work we find that the 30‐S binding of IF‐3 is rather sensitive to aurin‐tricarboxylic acid, a powerful inhibitor of polypeptide chain initiation, but relatively insensitive to streptomycin. IF‐3 binds weakly to both 23‐S and 16‐S ribosomal RNA and to 23‐S but not 43‐S ribosomal cores, Free IF‐3 readily exchanges with the 30‐S‐bound factor even at low temperature. There is also an exchange of 50‐S or 30‐S ribosomal subunits labelled with [3H]uracil into 70‐S ribosomes in the absence of the reactions of protein synthesis. This exchange is markedly temperature‐dependent and strongly inhibited at high (20 mM) Mg2+ concentrations.

Characterization of a viral messenger ribonucleoprotein particle accumulated during inhibition of polypeptide chain initiation in reovirus-infected L cells

A prior study has shown that L cells deprived of essential amino acids in isotonic Tris-buffered KCl show a marked and rapid inhibition of protein synthesis which is due to a decrease in the rate of initiation of new peptide chains. The inhibition, which is manifested by a disaggregation of polysomes with apparent conservation of mRNA, is rapidly reversed when the cells are resuspended in complete growth medium. This phenomenon has been applied in studies on the mechanism of mRNA storage and reutilization in reovirus-infected L cells. When reovirus-infected L cells are incubated in KCl-Tris medium, the viral messengers accumulate as ribonucleoprotein particles which sediment at approximately 50 S in sucrose density gradients and have a density in CsCl of 1.40. These 50-S messenger-containing ribonucleoprotein particles do not appear to be complexed with ribosomal subunits. The particles contain all 10 species of viral mRNA and have no demonstrable viral proteins. The mRNA from these particles is readily and rapidly mobilized into functional polysomes when the infected cells are restored to normal growth medium. Moreover, the mRNA moiety of partially purified 50-S ribonucleoprotein particles can direct the incorporation of amino acids into protein in a cell-free system.

An effect of pactamycin on the initiation of protein synthesis in reticulocytes

Low levels of pactamycin (about 10 −6 M) stop polypeptide synthesis by intact reticulocytes and their lysates after a lag of about two minutes. This effect which is similar to that of poly A, an inhibitor of polypeptide chain initiation, is not additive to that of poly A. Cells which have been pretreated with NaF and must form new polypeptide chains are more sensitive to pactamycin than cells which are able to complete pre-existing chains. High levels of pactamycin (⩾10 −5 M) inhibit elongation in addition to initiation. 3H-pactamycin binds to 80S ribosomes and the 40S subunits at 0°. Monosomes produced by NaF treatment of reticulocytes bind 3H-pactamycin very effectively, whereas those produced by RNase treatment of polyribosomes do not.