Release and recycling of eukaryotic initiation factor 2 in the formation of an 80 S ribosomal polypeptide chain initiation complex

Amitabha Chakrabarti, Umadas Maitra

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

The eukaryotic initiation factor (eIF)-5 mediates hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. The eIF-2•GDP formed under these conditions is released from the 40 S ribosomal subunit while initiator Met-tRNAf remains bound. The released eIF-2•GDP can participate in an eIF-2B-catalyzed GDP/GTP exchange reaction to reform the Met-tRNAf•eIF-2•GTP ternary complex. In contrast, when 60 S ribosomal subunits were also present in an eIF-5-catalyzed reaction, the eIF-2•GDP produced remained bound to the 60 S ribosomal subunit of the 80 S initiation complex. When such an 80 S initiation complex, containing bound eIF-2•GDP, was incubated with GTP and eIF-2B, GDP was released. However, eIF-2 still remained bound to the ribosomes and was unable to form a Met-tRNAf•eIF-2•GTP ternary complex. In contrast, when 60 S ribosomal subunits were preincubated with either free eIF-2 or with eIF-2•eIF-2B complex and then added to a reaction containing both the 40 S initiation complex and eIF-5, the eIF-2•GDP produced did not bind to the 60 S ribosomal subunits but was released from the ribosomes. Thus, the 80 S initiation complex formed under these conditions did not contain bound eIF-2•GDP. Under similar experimental conditions, preincubation of 60 S ribosomal subunits with purified eIF-2B (free of eIF-2) failed to cause release of eIF-2•GDP from the ribosomal initiation complex. These results suggest that 60 S ribosome-bound eIF-2•GDP does not act as a direct substrate for eIF-2B-mediated release of eIF-2 from ribosomes. Rather, the affinity of 60 S ribosomal subunits for either eIF-2, or the eIF-2 moiety of the eIF-2•eIF-2B complex, prevents association of 60 S ribosomal subunits with eIF-2•GDP formed in the initiation reaction. This ensures release of eIF-2 from ribosomes following hydrolysis of GTP bound to the 40 S initiation complex.

Original languageEnglish (US)
Pages (from-to)12964-12972
Number of pages9
JournalJournal of Biological Chemistry
Volume267
Issue number18
StatePublished - Jun 25 1992

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Eukaryotic Initiation Factor-2
Eukaryotic Initiation Factors
Ribosome Subunits
Eukaryotic Initiation Factor-2B
Recycling
Eukaryotic Initiation Factor-5
Peptides
Ribosomes
Guanosine Triphosphate
Hydrolysis
Association reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Release and recycling of eukaryotic initiation factor 2 in the formation of an 80 S ribosomal polypeptide chain initiation complex. / Chakrabarti, Amitabha; Maitra, Umadas.

In: Journal of Biological Chemistry, Vol. 267, No. 18, 25.06.1992, p. 12964-12972.

Research output: Contribution to journalArticle

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abstract = "The eukaryotic initiation factor (eIF)-5 mediates hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. The eIF-2•GDP formed under these conditions is released from the 40 S ribosomal subunit while initiator Met-tRNAf remains bound. The released eIF-2•GDP can participate in an eIF-2B-catalyzed GDP/GTP exchange reaction to reform the Met-tRNAf•eIF-2•GTP ternary complex. In contrast, when 60 S ribosomal subunits were also present in an eIF-5-catalyzed reaction, the eIF-2•GDP produced remained bound to the 60 S ribosomal subunit of the 80 S initiation complex. When such an 80 S initiation complex, containing bound eIF-2•GDP, was incubated with GTP and eIF-2B, GDP was released. However, eIF-2 still remained bound to the ribosomes and was unable to form a Met-tRNAf•eIF-2•GTP ternary complex. In contrast, when 60 S ribosomal subunits were preincubated with either free eIF-2 or with eIF-2•eIF-2B complex and then added to a reaction containing both the 40 S initiation complex and eIF-5, the eIF-2•GDP produced did not bind to the 60 S ribosomal subunits but was released from the ribosomes. Thus, the 80 S initiation complex formed under these conditions did not contain bound eIF-2•GDP. Under similar experimental conditions, preincubation of 60 S ribosomal subunits with purified eIF-2B (free of eIF-2) failed to cause release of eIF-2•GDP from the ribosomal initiation complex. These results suggest that 60 S ribosome-bound eIF-2•GDP does not act as a direct substrate for eIF-2B-mediated release of eIF-2 from ribosomes. Rather, the affinity of 60 S ribosomal subunits for either eIF-2, or the eIF-2 moiety of the eIF-2•eIF-2B complex, prevents association of 60 S ribosomal subunits with eIF-2•GDP formed in the initiation reaction. This ensures release of eIF-2 from ribosomes following hydrolysis of GTP bound to the 40 S initiation complex.",
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N2 - The eukaryotic initiation factor (eIF)-5 mediates hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. The eIF-2•GDP formed under these conditions is released from the 40 S ribosomal subunit while initiator Met-tRNAf remains bound. The released eIF-2•GDP can participate in an eIF-2B-catalyzed GDP/GTP exchange reaction to reform the Met-tRNAf•eIF-2•GTP ternary complex. In contrast, when 60 S ribosomal subunits were also present in an eIF-5-catalyzed reaction, the eIF-2•GDP produced remained bound to the 60 S ribosomal subunit of the 80 S initiation complex. When such an 80 S initiation complex, containing bound eIF-2•GDP, was incubated with GTP and eIF-2B, GDP was released. However, eIF-2 still remained bound to the ribosomes and was unable to form a Met-tRNAf•eIF-2•GTP ternary complex. In contrast, when 60 S ribosomal subunits were preincubated with either free eIF-2 or with eIF-2•eIF-2B complex and then added to a reaction containing both the 40 S initiation complex and eIF-5, the eIF-2•GDP produced did not bind to the 60 S ribosomal subunits but was released from the ribosomes. Thus, the 80 S initiation complex formed under these conditions did not contain bound eIF-2•GDP. Under similar experimental conditions, preincubation of 60 S ribosomal subunits with purified eIF-2B (free of eIF-2) failed to cause release of eIF-2•GDP from the ribosomal initiation complex. These results suggest that 60 S ribosome-bound eIF-2•GDP does not act as a direct substrate for eIF-2B-mediated release of eIF-2 from ribosomes. Rather, the affinity of 60 S ribosomal subunits for either eIF-2, or the eIF-2 moiety of the eIF-2•eIF-2B complex, prevents association of 60 S ribosomal subunits with eIF-2•GDP formed in the initiation reaction. This ensures release of eIF-2 from ribosomes following hydrolysis of GTP bound to the 40 S initiation complex.

AB - The eukaryotic initiation factor (eIF)-5 mediates hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. The eIF-2•GDP formed under these conditions is released from the 40 S ribosomal subunit while initiator Met-tRNAf remains bound. The released eIF-2•GDP can participate in an eIF-2B-catalyzed GDP/GTP exchange reaction to reform the Met-tRNAf•eIF-2•GTP ternary complex. In contrast, when 60 S ribosomal subunits were also present in an eIF-5-catalyzed reaction, the eIF-2•GDP produced remained bound to the 60 S ribosomal subunit of the 80 S initiation complex. When such an 80 S initiation complex, containing bound eIF-2•GDP, was incubated with GTP and eIF-2B, GDP was released. However, eIF-2 still remained bound to the ribosomes and was unable to form a Met-tRNAf•eIF-2•GTP ternary complex. In contrast, when 60 S ribosomal subunits were preincubated with either free eIF-2 or with eIF-2•eIF-2B complex and then added to a reaction containing both the 40 S initiation complex and eIF-5, the eIF-2•GDP produced did not bind to the 60 S ribosomal subunits but was released from the ribosomes. Thus, the 80 S initiation complex formed under these conditions did not contain bound eIF-2•GDP. Under similar experimental conditions, preincubation of 60 S ribosomal subunits with purified eIF-2B (free of eIF-2) failed to cause release of eIF-2•GDP from the ribosomal initiation complex. These results suggest that 60 S ribosome-bound eIF-2•GDP does not act as a direct substrate for eIF-2B-mediated release of eIF-2 from ribosomes. Rather, the affinity of 60 S ribosomal subunits for either eIF-2, or the eIF-2 moiety of the eIF-2•eIF-2B complex, prevents association of 60 S ribosomal subunits with eIF-2•GDP formed in the initiation reaction. This ensures release of eIF-2 from ribosomes following hydrolysis of GTP bound to the 40 S initiation complex.

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