A wheat germ cap-site factor functional in protein chain initiation

Abstract

Component C1 from wheat germ, a factor that functions in attaching ribosomes to mRNA, has been resolved into a fraction that does not bind to m7GDP-agarose (referred to as eIF4B) and one that binds and is eluted specifically by m7GDP. Both components are required for the attachment of ribosomes to [3H]methyl-labeled reovirus RNA and for the translation of a number of mRNAs, including the noncapped RNA of satellite tobacco necrosis virus. The component that binds to m7GDP-agarose, referred to as CSF (cap-site factor), contains primarily proteins of Mr 24,000, 26,000, and 75,000. Crosslinking studies with oxidized [3H]methyl-labeled reovirus RNA show that one of the lower molecular weight polypeptides of CSF interacts specifically with the 5'-cap of the mRNA in the absence of any other components. Incubation of component C1 and eIF4A in the presence of ATP results in the additional crosslinking of a 51- and a 65-kDa protein. In the absence of eIF4A, there is only the crosslinking of the lower molecular mass polypeptide (24 or 26 kDa). Attempts to reconstitute the C1 reaction with CSF and eIF4B result in a considerably diminished reaction. Crosslinking of eIF4A, however, is obtained in an incubation containing only CSF and eIF4A, suggesting that CSF may bring about an initial interaction of eIF4A with the 5' end of the mRNA.