Mechanism of polypeptide chain initiation in eukaryotes and its control by phosphorylation of the alpha subunit of initiation factor 2.

John Siekierka, L. Mauser, S. Ochoa

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Abstract

Earlier, we isolated eukaryotic initiation factor 2 (eIF-2)-stimulating protein (SP) as a homogeneous complex with eIF-2 (eIF-2-SP) and showed that, in the presence of Mg2+, eIF-2-SP promotes formation of a ternary complex with GTP and eukaryotic initiator methionyl tRNA (Met-tRNAi) (eIF-2-GTP-Met-tRNAi) catalytically. We now show that SP-bound eIF-2 exchanges with eIF-2 (eIF-2 exchange). Furthermore, in the presence of Mg2+, eIF-2-SP catalyzes the exchange of eIF-2-bound [3H]GDP with unlabeled GDP or GTP (GDP exchange) and the release of [3H]GDP when the ternary complex is formed from eIF-2-[3H]GDP, GTP, and [35S]Met-tRNAi. All these reactions are blocked by alpha-subunit, but not by beta-subunit, phosphorylation of eIF-2. The eIF-2 and GDP exchanges are compatible with the reaction eIF-2-GDP + SP in equilibrium EIF-2-SP + GDP reminiscent of the exchange between the Tu and Ts components of prokaryotic elongation factor 1 (EF-Tu and EF-Ts, respectively) EF-Tu-GDP + EF-Ts in equilibrium EF-Tu-EF-Ts + GDP. Due to the high affinity of GDP (approximately 100 times greater than that of GDP) for eIF-2, 40S (eIF-2-GTP-Met-tRNAi-40S) to 80S (Met-tRNAi-mRNA-80S) initiation complex conversion, which is accompanied by GTP hydrolysis, probably releases eIF-2 as eIF-2-GDP. Our results suggest that, in the presence of Mg2+, GDP binding restricts the availability of eIF-2 for chain initiation and that SP relieves this restriction in a catalytic fashion, provided that the alpha subunit of eIF-2 is not phosphorylated.

Original languageEnglish
Pages (from-to)2537-2540
Number of pages4
JournalProceedings of the National Academy of Sciences of the United States of America
Volume79
Issue number8
DOIs
StatePublished - 1 Jan 1982

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Prokaryotic Initiation Factor-2
Eukaryotic Initiation Factor-2
Eukaryota
Phosphorylation
Peptides
Guanosine Triphosphate
Peptide Elongation Factor Tu
Proteins

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title = "Mechanism of polypeptide chain initiation in eukaryotes and its control by phosphorylation of the alpha subunit of initiation factor 2.",
abstract = "Earlier, we isolated eukaryotic initiation factor 2 (eIF-2)-stimulating protein (SP) as a homogeneous complex with eIF-2 (eIF-2-SP) and showed that, in the presence of Mg2+, eIF-2-SP promotes formation of a ternary complex with GTP and eukaryotic initiator methionyl tRNA (Met-tRNAi) (eIF-2-GTP-Met-tRNAi) catalytically. We now show that SP-bound eIF-2 exchanges with eIF-2 (eIF-2 exchange). Furthermore, in the presence of Mg2+, eIF-2-SP catalyzes the exchange of eIF-2-bound [3H]GDP with unlabeled GDP or GTP (GDP exchange) and the release of [3H]GDP when the ternary complex is formed from eIF-2-[3H]GDP, GTP, and [35S]Met-tRNAi. All these reactions are blocked by alpha-subunit, but not by beta-subunit, phosphorylation of eIF-2. The eIF-2 and GDP exchanges are compatible with the reaction eIF-2-GDP + SP in equilibrium EIF-2-SP + GDP reminiscent of the exchange between the Tu and Ts components of prokaryotic elongation factor 1 (EF-Tu and EF-Ts, respectively) EF-Tu-GDP + EF-Ts in equilibrium EF-Tu-EF-Ts + GDP. Due to the high affinity of GDP (approximately 100 times greater than that of GDP) for eIF-2, 40S (eIF-2-GTP-Met-tRNAi-40S) to 80S (Met-tRNAi-mRNA-80S) initiation complex conversion, which is accompanied by GTP hydrolysis, probably releases eIF-2 as eIF-2-GDP. Our results suggest that, in the presence of Mg2+, GDP binding restricts the availability of eIF-2 for chain initiation and that SP relieves this restriction in a catalytic fashion, provided that the alpha subunit of eIF-2 is not phosphorylated.",
author = "John Siekierka and L. Mauser and S. Ochoa",
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Mechanism of polypeptide chain initiation in eukaryotes and its control by phosphorylation of the alpha subunit of initiation factor 2. / Siekierka, John; Mauser, L.; Ochoa, S.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 79, No. 8, 01.01.1982, p. 2537-2540.

Research output: Contribution to journalArticleResearchpeer-review

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N2 - Earlier, we isolated eukaryotic initiation factor 2 (eIF-2)-stimulating protein (SP) as a homogeneous complex with eIF-2 (eIF-2-SP) and showed that, in the presence of Mg2+, eIF-2-SP promotes formation of a ternary complex with GTP and eukaryotic initiator methionyl tRNA (Met-tRNAi) (eIF-2-GTP-Met-tRNAi) catalytically. We now show that SP-bound eIF-2 exchanges with eIF-2 (eIF-2 exchange). Furthermore, in the presence of Mg2+, eIF-2-SP catalyzes the exchange of eIF-2-bound [3H]GDP with unlabeled GDP or GTP (GDP exchange) and the release of [3H]GDP when the ternary complex is formed from eIF-2-[3H]GDP, GTP, and [35S]Met-tRNAi. All these reactions are blocked by alpha-subunit, but not by beta-subunit, phosphorylation of eIF-2. The eIF-2 and GDP exchanges are compatible with the reaction eIF-2-GDP + SP in equilibrium EIF-2-SP + GDP reminiscent of the exchange between the Tu and Ts components of prokaryotic elongation factor 1 (EF-Tu and EF-Ts, respectively) EF-Tu-GDP + EF-Ts in equilibrium EF-Tu-EF-Ts + GDP. Due to the high affinity of GDP (approximately 100 times greater than that of GDP) for eIF-2, 40S (eIF-2-GTP-Met-tRNAi-40S) to 80S (Met-tRNAi-mRNA-80S) initiation complex conversion, which is accompanied by GTP hydrolysis, probably releases eIF-2 as eIF-2-GDP. Our results suggest that, in the presence of Mg2+, GDP binding restricts the availability of eIF-2 for chain initiation and that SP relieves this restriction in a catalytic fashion, provided that the alpha subunit of eIF-2 is not phosphorylated.

AB - Earlier, we isolated eukaryotic initiation factor 2 (eIF-2)-stimulating protein (SP) as a homogeneous complex with eIF-2 (eIF-2-SP) and showed that, in the presence of Mg2+, eIF-2-SP promotes formation of a ternary complex with GTP and eukaryotic initiator methionyl tRNA (Met-tRNAi) (eIF-2-GTP-Met-tRNAi) catalytically. We now show that SP-bound eIF-2 exchanges with eIF-2 (eIF-2 exchange). Furthermore, in the presence of Mg2+, eIF-2-SP catalyzes the exchange of eIF-2-bound [3H]GDP with unlabeled GDP or GTP (GDP exchange) and the release of [3H]GDP when the ternary complex is formed from eIF-2-[3H]GDP, GTP, and [35S]Met-tRNAi. All these reactions are blocked by alpha-subunit, but not by beta-subunit, phosphorylation of eIF-2. The eIF-2 and GDP exchanges are compatible with the reaction eIF-2-GDP + SP in equilibrium EIF-2-SP + GDP reminiscent of the exchange between the Tu and Ts components of prokaryotic elongation factor 1 (EF-Tu and EF-Ts, respectively) EF-Tu-GDP + EF-Ts in equilibrium EF-Tu-EF-Ts + GDP. Due to the high affinity of GDP (approximately 100 times greater than that of GDP) for eIF-2, 40S (eIF-2-GTP-Met-tRNAi-40S) to 80S (Met-tRNAi-mRNA-80S) initiation complex conversion, which is accompanied by GTP hydrolysis, probably releases eIF-2 as eIF-2-GDP. Our results suggest that, in the presence of Mg2+, GDP binding restricts the availability of eIF-2 for chain initiation and that SP relieves this restriction in a catalytic fashion, provided that the alpha subunit of eIF-2 is not phosphorylated.

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