Protein synthesis in rabbit reticulocytes XIX: EIF-2 promotes dissociation of Met-tRNAf·EIF-1·GTP complex and Met-tRNAf binding to 40S ribosomes

Majumdar, Angshuman; Roy, R.; Das, Abhimanyu; Dasgupta, Asim; Gupta, Naba K.

doi:10.1016/0006-291x(77)91235-9

Cited by 41 publications

(13 citation statements)

References 15 publications

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“…The kinetics of inhibition of protein synthesis produced by aliquots of GSSG-treated S150 extracts were biphasic, and in- Hemin-supplemented S150 extracts were incubated as described for 30 hibition was greater than that attributable to any residual GSSG in the S150 aliquot after the activation of the inhibitor (Fig. la).…”

Section: Methodsmentioning

confidence: 99%

“…This phosphorylation of eIF-2 prevents the binding of Met-tRNAf to the 40S ribosomal subunit in crude and in partially purified systems (24,(26)(27)(28). Recent evidence indicates that the formation of the ternary complex [eIF-2-Met-tRNAf-GTP] and its subsequent binding to 40S subunits may involve other factors in addition to eIF-2 (29)(30)(31)(32) and that the phosphorylation of eIF-2 may inhibit its interaction with these factors (28,32).…”

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confidence: 99%

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Inhibition of protein synthesis initiation by oxidized glutathione: Activation of a protein kinase that phosphorylates the α subunit of eukaryotic initiation factor 2

Ernst

Levin

London

1978

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

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Oxidized glutathione (GSSG) (0.02-0.5 mM) inhibits reticulocyte lysates by a mechanism similar to that observed in heme deficiency. Incubation of hemin-supplemented postribosomal supernates with GSSG results in the activation of a translational inhibitor [I(GSSG)J. The activation of I(GSSG) is enhanced by the presence of an energy-regenerating system. The simultaneous addition of 1 mM dithiothreitol blocks the activation of the GSSG-induced inhibitor; however, once inhibitor is formed, its activity is not affected by 1 mM dithiothreitol. GSSG-treated postribosomal supernates and partially purified preparations of I(GSSG) inhibit protein synthesis in emin-supplemented lysates with biphasic kinetics. Inhibition by I(GSSG) is blocked by cyclic AMP (2-10 mM) and is potentiated by ATP (2 mM). The inhibition is also blocked or reversed by eukaryotic initiation factor eIF-2. The activation of I(GSSG) is accompanied by an increased cyclic AMP-independent protein kinase activity which phosphorylates the 38,000da ton component (a subunit) of eIF-2; however, GSSG treatment of supernates does not alter the activity of the cyclic AMP-independent protein kinase activity that phosphorylates the 49,000-dalton polypeptide component (ft subunit) of eIF-2.These data indicate that GSSG treatment of reticulocyte lysates results in the activation of a protein kinase with inhibitory and phosphorylation properties similar to those of the heme-regulated cyclic AMP-independent protein kinase which is activated in heme deficiency.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Inhibition of protein synthesis initiation by oxidized glutathione: Activation of a protein kinase that phosphorylates the α subunit of eukaryotic initiation factor 2

Ernst

Levin

London

1978

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

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“…These observations are explained by the results reported here. Our results provide a logical explanation for the so-called ternary complex dissociation reaction (15,16). Ternary complex dissociation has been believed to be promoted by a ternary complex dissociation factor (TDF), later renamed CeeIF-2B, present in crude reticulocyte lysate fractions.…”

Section: Resultsmentioning

confidence: 58%

“…CoeIF-2B was further believed to be required for 40S complex formation, a paradoxical requirement, for it is generally accepted that the 40S complex is formed by interaction of the ternary complex (eIF-2-GTP-MettRNAi) with a 40S ribosomal subunit. We reported previously (11) that, under the conditions of Majumdar et al (15), ternary complex dissociation is promoted by virtually homogeneous eIF-2'SP and cast doubt on the existence ofa dissociating factor (CoeIF-2B) distinct from eIF-2-SP. In this connection, it may be noted that the molecular weight of CoeIF-2B is given as 450,000 (15), which is the same reported for eIF-2'SP (6,8).…”

Section: Resultsmentioning

confidence: 91%

“…We reported previously (11) that, under the conditions of Majumdar et al (15), ternary complex dissociation is promoted by virtually homogeneous eIF-2'SP and cast doubt on the existence ofa dissociating factor (CoeIF-2B) distinct from eIF-2-SP. In this connection, it may be noted that the molecular weight of CoeIF-2B is given as 450,000 (15), which is the same reported for eIF-2'SP (6,8). It is now evident that, in our earlier work on ternary complex dissociation (11), we were in fact dealing with reversibility of ternary complex formation.…”

Section: Resultsmentioning

confidence: 91%

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Polypeptide chain initiation in eukaryotes: reversibility of the ternary complex-forming reaction.

Siekierka¹,

Manne²,

Mauser³

et al. 1983

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

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In the last step of polypeptide chain initiation in eukaryotes, the interaction of the 40S preinitiation complex eIF-2 GTP Met-tRNAj 4OS [the complex between the 40S ribosomal subunit and the ternary complex containing equimolar amounts of eukaryotic initiation factor 2 (eIF-2), GTP, and eukaryotic initiator methionyl tRNA (Met-tRNA;)] with a 60S ribosomal subunit in the presence of mRNA, cap binding protein (with "capped" messengers), ATP, and the initiation factors eIF-3, eIF-4a, -4b, -4c, and eIF-5, results in the formation of an 80S initiation complex (MettRNAj'80S'mRNA) with concomitant hydrolysis of GTP and liberation of eIF-2 for recycling in subsequent initiation events. However, at physiological Mg2+ concentrations, GDP is known to have '100-fold greater affinity than GTP for eIF-2 and eIF-2 is believed to be released in the form of an eIF-2'GDP complex. Previously, we have shown that initiation factor SP (for eIF-2-stimulating protein) promotes the exchange of eIF-2-bound GDP for GTP and catalyzes ternary complex formation in the presence of Met-tRNA;. Binding of GDP by eIF-2 is indeed so tight that, as we now show, homogeneous preparations of eIF-2 contain upward of 0.5 mol of GDP/mol of eIF-2. We further show that, in the presence of Mg2+ and catalytic amounts of SP, ternary complex formation conforms to the overall reversible reaction eIF-2'GDP + GTP + Met-tRNA, = eIF-2 GTP'Met-tRNA; + GDP.The first step of polypeptide chain initiation in eukaryotes is the formation of a ternary complex containing equimolar amounts of eukaryotic initiation factor 2 (eIF-2), GTP, and eukaryotic initiator methionyl tRNA (Met-tRNAi). This complex binds to a 40S ribosomal subunit, giving rise to a 40S preinitiation complex. In the presence of a 60S ribosomal subunit, mRNA, additional factors, and ATP, an 80S initiation complex is formed, setting the stage for chain elongation. Formation of the 80S complex is accompanied by GTP hydrolysis and eIF-2 release (see the diagram in ref. 1). Ternary complex formation is inhibited by Mg2+ (2), which, however, is required for the ensuing steps (3). In the presence of Mg2+, eIF-2 has at least 100-fold higher affinity for GDP than for GTP (4), suggesting that the factor is released as an eIF-2-GDP complex on completion of each initiation round. In the presence of Mg2+, formation of a ternary complex (which contains GTP) requires, therefore, a mechanism for exchange of eIF-2-bound GDP for GTP (5, 6). This exchange is catalyzed by a factor termed SP [for eIF-2-stimulating protein (6)] or GEF [for GDP exchange (7)]. The strong similarity of ternary complex formation in eukaryotic chain initiation and prokaryotic chain elongation has been pointed out (6). It is now apparent that eIF-2 and SP are the counterparts of the chain elongation factors EF-Tu and EF-Ts, respectively (6). GDP (unpublished data), providing direct evidence for reversal of reaction a. In this paper, we show that, unlike eIF-2'SP, eIF-2 is isolated in the form of an eIF-2-GDP complex and present evidence for...

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Regulation of Eukaryotic Translation

Kaempfer

1984

Viral Cytopathology

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Protein synthesis in rabbit reticulocytes XIX: EIF-2 promotes dissociation of Met-tRNAf·EIF-1·GTP complex and Met-tRNAf binding to 40S ribosomes

Cited by 41 publications

References 15 publications

Inhibition of protein synthesis initiation by oxidized glutathione: Activation of a protein kinase that phosphorylates the α subunit of eukaryotic initiation factor 2

Inhibition of protein synthesis initiation by oxidized glutathione: Activation of a protein kinase that phosphorylates the α subunit of eukaryotic initiation factor 2

Polypeptide chain initiation in eukaryotes: reversibility of the ternary complex-forming reaction.

Regulation of Eukaryotic Translation

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