Evidence that phosphorylation of eIF-2(alpha) prevents the eIF-2B-mediated dissociation of eIF-2 X GDP from the 60 S subunit of complete initiation complexes.

M Gross,M Wing,C Rundquist,M S Rubino

doi:10.1016/s0021-9258(18)48329-9

Abstract

Recent observations have indicated that eukaryotic initiation factor (eIF)-2 and GTP or GDP normally bind to 60 S ribosomal subunits in rabbit reticulocyte lysate and that when eIF-2 alpha is phosphorylated and polypeptide chain initiation is inhibited, eIF-2 X GDP accumulates on 60 S subunits due to impaired dissociation that is normally mediated by the reversing factor (eIF-2B). Current findings now indicate that inhibition due to phosphorylation of eIF-2 alpha is mediated, at least in part, by the inability to dissociate eIF-2 X GDP from the 60 S subunit of complete initiation complexes. At the onset of inhibition, there is an accumulation of Met-tRNA(f) and eIF-2 on the polysomes, despite a marked reduction in Met-tRNA(f) bound to 40 S subunits and Met-peptidyl-tRNA bound to the polysomes. This initial effect is not associated with the formation of "half-mers" (polysomes containing an extra unpaired 40 S subunit), and the 40 S X Met-tRNA(f) complexes, though reduced, still sediment at 43 S. When inhibition is maximal and the polysomes are largely disaggregated, there is an accumulation of 48 S complexes consisting of a 40 S subunit and Met-tRNA(f) bound to globin mRNA as well as small polysomal half-mers, such that residual protein synthesis occurs to about the same degree on "1 1/2"s and "2 1/2"s as on mono-, di-, and triribosomes. Exogenous eIF-2B increases protein synthesis on mono-, di-, and triribosomes and decreases that on half-mers. This is associated with reduced binding of Met-tRNA(f) and eIF-2 to ribosomal particles sedimenting at 80 S and greater and a shift from 48 S to 43 S complexes. These results suggest that eIF-2B must normally promote dissociation of eIF-2 X GDP from the 60 S subunit of complete initiation complexes before they can elongate but cannot when eIF-2 alpha is phosphorylated, resulting in the accumulation of these complexes, some of which dissociate into Met-tRNA(f) X 40 S X mRNA and 60 S X eIF-2 X GDP.

Highlights

These studies have suggested that the inhibithe onset of inhibition, there is acncumulation of Met- tion due to phosphorylation is mediated entirely at the level tRNAf and eIF-2 on the polysomes, despite a marked of soluble reactions and only secondarily involves ribosomal reduction in Met-tRNAf bound to 40 S subunits and components, our earlier observation [22,23] that phosphoryl
Maximal and thpeolysomes are largely disaggregated, We [24] and Thomaset al. [25] have recently reported that there is an accumulation of 48 S complexes consisting eIF-2and GTP or GDP normally bind to 60 S ribosomal of a 40 S subunitand Met-tRNArbound to globin subunits, that upon phosphorylation of eIF-Pa there is inmRNA as well as small polysomal half-mers, suchthat creased binding of eIF-2-GDP and eIF-2(a-P).GDP t6o0 S
Fig. 3illustrates the steady-state labeling of ribosomal large pool of run-off ribosomes generated in these samples, particles in lysate incubated in the absence of hemin or presence of hemin plus 0.1 pg/ml poly(1.C) and with either [35S]Met-tRNAfor [3H]Ala-tRNA.The results indicate that when inhibition due to phosphorylation of eIF-2a is maximal since binding to diribosomes shows the same pattern as that on 80 S particles and since a separate inhibitor of initiation, thecap analogue 7MeGTP, does not promote appreciable accumulation of Met-tRNAf on mono- and polyribosomes in both [35S]methionine and [3H]alanine label are associated with “11h”sand ‘‘2%”~ waesll as monoribosomesand disomes

Summary

Introduction

Fig. 3illustrates the steady-state labeling of ribosomal large pool of run-off ribosomes generated in these samples, particles in lysate incubated in the absence of hemin or presence of hemin plus 0.1 pg/ml poly(1.C) and with either [35S]Met-tRNAfor [3H]Ala-tRNA.The results indicate that when inhibition due to phosphorylation of eIF-2a is maximal since binding to diribosomes shows the same pattern as that on 80 S particles and since a separate inhibitor of initiation, thecap analogue 7MeGTP, does not promote appreciable accumulation of Met-tRNAf on mono- and polyribosomes in both [35S]methionine and [3H]alanine label are associated with “11h”sand ‘‘2%”~ waesll as monoribosomesand disomes.

Results

Conclusion