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

A Chakrabarti,U Maitra

doi:10.1016/s0021-9258(18)92807-3

A Chakrabarti, U Maitra

Open Access

https://doi.org/10.1016/s0021-9258(18)92807-3

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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.

Highlights

Eukaryotic initiation factor[5], isolated from Eukaryotic initiation factor 5 was initially isolated in sevrabbit reticulocyte lysates, is a monomeric protein of eral laboratories from a crude mixture of the 0.5 M KC1-wash 58-62 kDa
This system consisted of a mixture of defined ttuwShhn*aeeiAsts4ruU.a0nAGpaSinf*dfaiMenlcyaiettsneitiad*dsttoibRofqynNuetIaAhcFnoe-ftm5*itp-eapcrItlaeFievtsx-aee2.lnyTchzGeheyoeTddfPrr6oar)0eltyeawSoscofiitrsftiGihobtnThoeisPsIopFbmrr-oeo5aaudlcnustrdciueobtbtssnyou- lteaai1demtsaionioinnndnofaEtahccFyet-ol2f-rut,sRn,aNcentIAdiFo-snp2,r,Aoo' fetTeeIPiFanc,3hGf,roTaefcIPFtt,ih-o4entAhsis,etocewloIaFont-ete4adliBonin,nigniatagintatidhoteienoIFnfoa-t4fchaCtecor.troSsirntEsuitFdiin--gel filtration indicated that both eIF-2-GDP binary the sequential binding of initiator Met-tRNAf andmRNA to complex and Pi formed were released from the ribo- the small ribosomal subunit (40 S) and the joining of the somal complex whereas Met-tRNAfremained bound to larger ribosomal subunit (60 S) to form a functional 80 S 40 S ribosomes as a Met-tRNAr.[40]
EIF-5 was beled eIF-5 indicated that thisprotein was not associ- found to be essential for the joining of 60 S ribosomal subunits ated with the 40 S-AUG-Met-tRNArcomplex; similar to the 40 S initiation complex to form a functional 80 S

Summary

40 S subunits

Fractions of 0.4 ml were collected, and the cated an intense "'P-labeled phosphoprotein corresponding to a M , =. Radioactivity content in each fraction was measured by counting in 58,000 (data not shown).

RESULTS

Findings

DISCUSSION