Preparation and characterization of eukaryotic initiation factor EIF-3. Formation of binary (EIF-3-Met-tRNAf) and ternary (EIF-3-Met-tRNAf-GTP) complexes.
1976; Elsevier BV; Volume: 251; Issue: 7 Linguagem: Inglês
10.1016/s0021-9258(17)33636-0
ISSN1083-351X
AutoresRajinder Singh Ranu, Ira G. Wool,
Tópico(s)Bacteriophages and microbial interactions
ResumoThe 133,000 x g supernatant fraction prepared from ascites cells in 20 mM KC1 (low KC1 supernatant) contained the initiation factors EIF-1 and EIF-2 (and the elongation factors EF-1 and EF-2) but lacked EIF-3; thus, low KC1 supernatant could be used to assay for EIF-3.EIF-3 was prepared from a crude initiation factor preparation (a 250 mM KC1 extract of ascites cell ribosomes precipitated with 70% saturated ammonium sulfate) by chromatography on DEAE-Sephadex A-50 and hydroxylapatite.The EIF-3 had no detectable EIF-1 and little or no EIF-2.Factor EIF-3 was required for 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-tRNA,, and if GTP was present the factor formed a ternary complex (EIF-S.Met-tRNAl.GTP).The EIF-3 preparation had no methionyl-tRNA synthetase activity to account for binding.Complex.formation was with eukaryotic Met-tRNA, and no other aminoacyl-tRNA.The binary and ternary complexes were retained quantitatively on Millipore filters (which was tie 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 ratio of GTP to Met-tRNA, in the ternary complex appeared to be 1.The binary and the ternary complexes transferred Met-tRNA, to 40 S ribosomal subunits, but not to 60 S subparticles.The factor-dependent binding of Met-tRNA, 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 (Nakaya, K.
Referência(s)