Initiation of translation with native 40-S ribosomal subunits.

Abstract

We have studied the steps taking place in initiation of protein synthesis in rabbit reticulocyte cell-free systems by using native 40-S subunits. Binding of the initiator Met-tRNAf to the native 40-S subunits took place without additions of initiation factors, in the presence or absence of poly(A,G,U) but GTP is required. There is no requirement of mRNA for the binding reaction, suggesting the presence of eIF-2 and eIF-3 in native 40-S subunits. Polydextran sulphate and N-ethylmaleimide block the binding of Met-tRNAf to native 40-S subunits, whereas pactamycin and edeine A1 enhance the binding. Aurintricarboxylic acid inhibits the poly(A,G,U)-dependent stimulation of the reaction and releases some initiation factor(s) from native 40-S subunits. Addition of 60-S subunits to the native 40-S · Met-tRNAf complex in the absence of eIF-5 leads to a 80-S · Met-tRNAf complex, reactive with puromycin, suggesting that this initiation factor was present in the native 40-S subunits. Formation of methionyl-puromycin with the 80-S · Met-tRNAf complex is blocked by inhibitors of peptide bond formation, such as anisomycin, trichodermin, trichothecin, bruceantin and harringtonine. Pactamycin, edeine A1, aurintricarboxylic acid, pyrocatechol violet and adrenochrome do not inhibit peptide bond formation but lead to the formation of an unusual 40-S · Met-tRNAf complex which, after the joining of the 60-S subunit, is not reactive in the puromycin reaction.