DETERMINATION OF THE AMINO-ACID-SEQUENCE OF RABBIT, HUMAN, AND WHEAT-GERM PROTEIN-SYNTHESIS FACTOR EIF-4C BY CLONING AND CHEMICAL SEQUENCING

The small eukaryotic initiation factor (eIF)-4C is implicated in the i nitiation pathway, where it enhances ribosome dissociation into subuni ts and stabilizes the binding of the initiator Met-tRNA(i) to 40 S rib osomal subunits. In order to elucidate the function of eIF-4C, its str ucture has been further characterized. The amino acid sequence of many peptides from rabbit reticulocyte and wheat germ eIF-4C have been det ermined chemically. From the chemical sequencing of the rabbit protein , it was noted that at least two different eIF-4C molecules were prese nt which differed by conservative substitutions at three positions (2 aspartic acid for glutamic acid switches and 1 valine for isoleucine s witch). By the use of unique sequences with low codon degeneracy, prim ers were used to obtain a polymerase chain reaction product of appropr iate size and sequence. This product was then used to isolate full-len gth coding sequence cDNA clones for human eIF-4C. A similar strategy w as used to design PCR primers and then isolate a wheat cDNA clone whic h lacked the coding region for the first 23 amino acids, but contained a complete 3'-untranslated region. The protein amino acid sequence of wheat germ eIF-4C is 68% identical with the mammalian protein, and, a llowing for the most conservative substitutions, the proteins are 76% similar. Both the mammalian and wheat germ proteins are 143 amino acid s in length and have molecular weights of abo 16,400. A unique feature of eIF-4C is.its apparent ''polarity'' as 9 of the first 15 amino aci ds are basic while 13 of the last 20 amino acids are acidic. This dipo le.nature may enable the protein to interact with both the ribosome (p erhaps via the rRNA) and other translation initiation factors.