Interaction of Wheat Germ Protein Synthesis Initiation Factor eIF-(iso)4F and Its Subunits p28 and p86 with m7GTP and mRNA Analogues

Ma Sha,Yahong Wang,Ting Xiang,Ann Van Heerden,Karen S Browning,Dixie J Goss

doi:10.1074/jbc.270.50.29904

Abstract

The binding of p28, p86, and native wheat germ eIF-(iso)4F with m7GTP and oligonucleotides was measured and compared. The purified subunits (p28, 28 kDa and p86, 86 kDa) of wheat germ protein synthesis initiation factor eIF-(iso)4F have been obtained from Escherichia coli expression of the cloned DNA (van Heerden, A., and Browning, K. S. (1994) J. Biol. Chem. 269, 17454-17457). The binding of the 5'-terminal cap analogue m7GTP to the small subunit (p28) of eIF-(iso)4F as a function of pH, temperature, and ionic strength is described. The mode of binding of p28 to cap analogues is very similar to the intact protein. Assuming that all tryptophan residues contribute to p28 and eIF-(iso)4F fluorescence, iodide quenching shows that all 9 tryptophan residues in p28 are solvent-accessible, while only 6 out of 16 tryptophan residues are solvent-accessible on the intact eIF-(iso)4F. The fluorescence stopped-flow studies of eIF-(iso)4F and p28 with cap show a concentration-independent conformational change. The rate of this conformational change was approximately 10-fold faster for the isolated p28 compared with the native eIF-(iso)4F. From these studies it appears that cap recognition resides in the p28 subunit. However, p86 enhances the interaction with capped oligonucleotides and probably is involved in protein-protein interactions as well. Both subunits are required for helicase activity.

Highlights

Recognition of the m7-cap structure of mRNA by eucaryotic initiation factors and formation of 48 S initiation complex are important steps in initiation of protein synthesis in eucaryotic cells
One wheat germ factor is required for ATP hydrolysis and stimulation of protein synthesis in an eIF-4F or eIF-(iso)4F deficient translation system (Lax et al, 1985, 1986b)
Wheat germ eIF-(iso)4F can substitute for mammalian eIF-4F in an RNA-dependent ATPase activity and in cross-linking of mammalian eIF-4A to the cap of oxidized mRNA (Abramson et al, 1988)

Summary

MATERIALS AND METHODS

Buffer A, used for fluorescence measurements, consisted of 20 mM HEPES-KOH, 100 mM KCl, 1 mM dithiothreitol, and 1 mM MgCl2 and was adjusted to the appropriate pH. Buffer B, used in isolation of the wheat germ factors, consisted of 20 mM HEPES-KOH, pH 7.6, 0.1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, and KCl as indicated. The 40% ammonium sulfate fraction was loaded onto a DE52 column, equilibrated with buffer B containing 40 mM KCl (B-40). The double-stranded RNA (Tm ϭ 65 °C) for the helicase assay of eIF-(iso)4F was prepared by annealing the partially complementary III and IV single-stranded RNA. The unwinding reaction for the helicase assay was performed by incubating partially double-stranded RNA with eucaryotic initiation factors (5 ␮M eIF-4A, 2 ␮M p28, p86, and eIF-(iso)4F were used) in 20 mM Tris-HCl buffer (pH 7.5) containing 2 mM ATP, 1 mM magnesium acetate. After rapid mixing of the protein with the mRNA cap, the time course of the intrinsic fluorescence intensity was recorded

RESULTS

Initiation factors

DISCUSSION