Function of the wheat eukaryotic initiation factors eIF(iso)4G and eIF4B in translation

The translation initiation factor eIF(iso)4F consists of a small cap binding protein, eIF(iso)4E or p28, and a large subunit, eIF(iso)4G or p86. Domains of p86 were mapped by mutational analysis. The protein contains a specific N-terminal p28 binding site between amino acids 52 and 89. This region is required for p86 to interact with the cap-binding protein to form a complex active in supporting translation initiation. The C-terminal 300 amino acids of p86 are not required for translation.;The initiation factor eIF4A binds to p86, rather than p28, in associating with eIF(iso)4F. It appears likely that eIF4A binds to p86 at a domain located in the central region of p86, between amino acids 328-511. The eIF4A and RNA-dependent ATP-hydrolysis activity of eIF(iso)4F is located on p86, as p28 is not required for this activity. Loss of the putative eIF4-binding domain results in loss of ATP-hydrolysis activity as well as activity in the translation assay.;The p86 protein is phosphorylated by a kinase from wheat germ between amino acids 511-624, providing another piece of evidence that the C-terminal portion of p86 functions in regulation rather than in translation itself.;This work presents for the first time the entire gene of the wheat translation initiation factor eIF4B and demonstrates that it can be expressed in E. coli and purified as an active protein. Evidence is presented that the unphosphorylated recombinant form of eIF4B is at least as active as native eIF4B (which may be at least partially phosphorylated) in translation initiation in vitro. Phosphorylation of the protein with a kinase from wheat germ does not increase eIF4B activity. It is also shown that eIF4B self-associates to form a dimer, and that it may associate with eIF4A in the initiation complex.;This work is the first to demonstrate a difference between eIF4F and its isozyme form, eIF(iso)4F, in an assay associated with translation initiation. The eIF4F ATP-hydrolysis activity can be stimulated by eIF4B, while the ATP-hydrolysis activity of its isozyme form appears to be insensitive to eIF4B stimulation.