Function of eukaryotic initiation factor 5 in the formation of an 80 S ribosomal polypeptide chain initiation complex

Amitabha Chakrabarti, Umadas Maitra

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59 Scopus citations

Abstract

Eukaryotic initiation factor 5 (eIF-5), isolated from rabbit reticulocyte lysates, is a monomeric protein of 58-62 kDa. The function of eIF-5 in the formation of an 80 S polypeptide chain initiation complex from a 40 S initiation complex has been investigated. Incubation of the isolated 40 S initiation complex (40 S·AUG·Met·tRNAf·eIF-2 GTP) with eIF-5 resulted in the rapid and quantitative hydrolysis of GTP bound to the 40 S initiation complex. The rate of this reaction was unaffected by the presence of 60 S ribosomal subunits. Analysis of eIF-5-catalyzed reaction products by gel filtration indicated that both eIF-2·GDP binary complex and Pi formed were released from the ribosomal complex whereas Met-tRNAf remained bound to 40 S ribosomes as a Met-tRNAf·40 S·AUG complex. Reactions carried out with biologically active 32P-labeled eIF-5 indicated that this protein was not associated with the 40 S·AUG·Met-tRNAf complex; similar results were obtained by immunological methods using monospecific anti-eIF-5 antibodies. The isolated 40 S·AUG·Met-RNAf complex, free of eIF-2·GDP binary complex and eIF-5, readily interacted with 60 S ribosomal subunits in the absence of exogenously added eIF-5 to form the 80 S initiation complex capable of transferring Met-tRNAf into peptide linkages. These results indicate that the sole function of eIF-5 in the initiation of protein synthesis is to mediate hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. This leads to formation of the intermediate 40 S·AUG·Met-tRNAf and dissociation of the eIF-2·GDP binary complex. Subsequent joining of 60 S ribosomal subunits to the intermediate 40 S·AUG·Met-tRNAf complex does not require participation of eIF-5. Thus, the formation of an 80 S ribosomal polypeptide chain initiation complex from a 40 S ribosomal initiation complex can be summarized by the following sequence of partial reactions. (40 S·AUG·Met-tRNAf-eIF-2-GTP) →eIF-5 (40 S·AUG·Met-tRNAf) + (eIF-2·GDP) + Pi (1) (40 S·AUG·Met-tRNAf) + 60 S → (80 S·AUG·Met-tRNAf) (2) 80 S initiation complex.

Original languageEnglish (US)
Pages (from-to)14039-14045
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number21
StatePublished - 1991

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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