JBC, Vol. 251, Issue 7, 1926-1935, Apr, 1976
Preparation and characterization of eukaryotic initiation factor EIF-3. Formation of binary (EIF-3-Met-tRNAf) and ternary (EIF-3-Met-tRNAf-GTP) complexes
R. S. Ranu and I. G. Wool
The 133,000 X g supernatant fraction prepared from ascites cells in 20 mM
KCl (low CKl supernatant) contained the initiation factors EIF-1 and EIF-2
(and the elongation factore EF-1 and EF-2) but lacked EIF-3; thus, low KCl
supernatant could be used to assay for EIF-3. EIF-3 was prepared from a
crude initiation factor perparation (a 250 mM KCl extract of ascites cell
ribosomes precipitated with 70% saturated ammonium sulfate) by
chromatography on DEAE-Sephadex A-50 and hydroxylapatite. The EIF-O had no
detectable EIF-1 and little or no EIF-2. Factor EIF-3 was required fro
translation of encephalomyocarditis virus RNA. The molecular weight of
EIF-3 was estimated by Sephadex G-200 filtration to be 139,000; the
sedimentation coefficient was calculated to be about 5.8. EIF-3 formed a
binary complex specifically with the initiator tRNA, Met-tRNAf, and if GTP
was present the factor formed a ternary complex (EIF-3-Met-tRNAf-GTP). The
EIF-3 preparation had no methionyl-tRNA synthetase activity to account for
binding. Complex-formation was with eukaryotic Met-tRNAf and no other
aminoacyl-tRNA. The binary and ternary complexes were retained
quantitatively on Millipore filters (which was the most convenient assay),
but they could also be demonstrated by filtration through Sephadex G-100 or
by glycerol gradient centrifugation. GTP increased the rate, the amount,
and the stability of complex formed; the ration of GTP to Met-tRNAf in the
ternary complex appeared to be 1. The binary and the ternary complexes
transferred Met-tRNAf to the 40 S ribosomal subunits, but not to 60 S
subparticles. The factor-dependent binding of Met-tRNAf to the 40 S subunit
did not require mRNA (or GTP). In the presence of 60 S subunits, the
initiator tRNA bound to 40 S subunits was not transferred to 80 S ribosomes
even if mRNA was added--that reaction may require another initiation
factor. Treatment of EIF-3 with N-ethylmaleimide led to loss of its
activity in complex formation and in support of the translation of
encephalomyocarditis virus RNA. In addition to forming the binary and
ternary complexes, and supporting the translation of encephalomyocarditis
virus RNA, EIF-3 also increases the number of free ribosomal subunits by
either preventing their association or causing dissociation of 80 S
couples.
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