Inhibition of protein synthesis by the heme-controlled eIF-2 alpha kinase leads to the appearance of mRNA-containing 48S complexes that contain eIF-4E but lack methionyl-tRNA(f).
Phosphorylation of the initiation factor eIF-2 by the heme-regulated eIF-2 alpha kinase (HCR) results in pronounced inhibition of protein synthesis and of binding of Met-tRNA(f) to 40S subunits in reticulocyte lysates. This inhibition is associated with the appearance of a more rapidly sedimenting 48S complex; this contains mRNA detectable by poly(U) hybridization, but not Met-tRNA(f). In contrast, 48S complexes that accumulate in the presence of the initiation inhibitor edeine contain both Met-tRNA(f) and mRNA. We have compared the composition of the particles that accumulate in the presence of HCR with those seen in the presence of edeine and find that both particles contain the cap binding protein, eIF-4E. Moreover, both particles exhibit a buoyant density of 1.40 g/ml in CsCl equilibrium density gradients. This is consistent with the presence of 500-700 kDa of protein additional to ribosomal structural protein, and suggests the presence of eIF-3 on both types of 48S complex. Lysates pre-treated with HCR and then treated with edeine show the ability to accumulate 48S complexes containing Met-tRNA(f), though at a slower rate than control lysates. These observations are discussed in the light of mechanisms previously suggested for the appearance of 48S particles following HCR treatment. In addition, we have observed association of eIF-4E with polysomes and 80S monosomes in reticulocyte lysates, suggesting that this factor may not be released immediately following the binding of the 40S ribosomal subunit to the 5' end of the mRNA.
Joshi
B
B
School of Biological Sciences, University of Sussex, Brighton, England.
Morley
S J
SJ
Rhoads
R E
RE
Pain
V M
VM
D000818:Animals; D002499:Centrifugation, Density Gradient; D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors / Q000378:metabolism*; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis*; D012358:RNA, Transfer, Met / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D019892:eIF-2 Kinase
D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase