In vitro synthesis, phosphorylation, and localization on 48 S initiation complexes of human protein synthesis initiation factor 4E.
Complementary DNA for human eukaryotic initiation factor 4E (eIF-4E) was transcribed in vitro and the transcripts used to direct protein synthesis in a cell-free reticulocyte translation system. The predominant translation product was 25 kDa, was bound to a m7GTP-Sepharose affinity column, and was specifically eluted with m7GTP. Both phosphorylated (P) and unphosphorylated (U) forms of eIF-4E were synthesized, and the P/U ratio increased as a function of incubation time in the reticulocyte lysate system. Both forms were quantitatively retained on m7GTP-Sepharose. When translation reactions were resolved on sucrose density gradients, the 35S-labeled eIF-4E sedimented predominantly at 3-4 S. However, in the presence of edeine or guanylyl imidodiphosphate, both of which cause accumulation of 48 S initiation complexes, eIF-4E was detected in the 48 S region. In the presence of sparsomycin, used to accumulate 80 S initiation complexes, no eIF-4E was observed in the 80 S region. No change in the eIF-4E distribution was caused by m7GTP. These results are consistent with a model whereby eIF-4E is transferred to the 43 S initiation complex together with mRNA and is released from the initiation complex when the 60 S ribosomal subunit joins.
Hiremath
L S
LS
Department of Biochemistry, College of Medicine, University of Kentucky, Lexington 40536-0084.
Hiremath
S T
ST
Rychlik
W
W
Joshi
S
S
Domier
L L
LL
Rhoads
R E
RE
D000818:Animals; D001483:Base Sequence; D039561:Eukaryotic Initiation Factor-4E; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure; D013698:Templates, Genetic; D014158:Transcription, Genetic
D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D012333:RNA, Messenger