Protein Synthesis Initiation in Eukaryotes

Abstract

Abstract A stepwise assembly of the eukaryotic 80 S initiation complex [80 S, Met-tRNAf, mRNA] was achieved at 1.5 to 3 mm MgCI2 utilizing ribosomal factors and stripped 40 S and 60 S ribosomal subunits from mouse fibroblasts (L cells), rat liver Met-tRNAf, and reovirus mRNA. Chromatography on DEAE-cellulose of a ribosomal wash from L cells yields three initiation factors (IF-L1, IF-L2, IF-L3) which mediate separate steps of the protein initiation sequence. IF-L3 contains the Met-tRNAf binding factor; IF-L2 functions as a mRNA insertion factor. The initiation sequence proceeds through at least four steps. Step 1, IF-L3 binds Met-tRNAf and GTP to form a ternary complex [IF-L3, Met-tRNAf, GTP] in the absence of ribosomes, mRNA, and MgCl2. Step 2, at 1.5 mm MgCl2, the ternary complex binds to the stripped 40 S subunit to form a [40 S, Met-tRNAf] complex, a reaction which is enhanced 3- to 5-fold by IF-L1. Step 3, at 1.5 mm MgCl2 in the presence of IF-L2, the [40 S, Met-tRNAf] complex binds reovirus mRNA to form a [40 S, Met-tRNAf, mRNA] complex. Step 4, at 3 mm MgCl2, the 60 S ribosomal subunit associates with the [40 S, Met-tRNAf, mRNA] complex to form the [80 S, Met-tRNAf, mRNA] initiation complex; IF-L1 significantly increases 80 S complex formation when IF-L2 and IF-L3 are limiting. The initiation complex formed in the sequential system is functional, and under appropriate conditions transfers its methionine to methionyl-puromycin or polypeptide. The sequence requires GTP; β,γ-methylene guanosine 5'-triphosphate replaces GTP in the first three steps, but not in the final step. Addition of 50 µm pactamycin selectively inhibits formation of the [80 S, Met-tRNAf, mRNA] complex in Step 4; other steps in the sequence are unaffected.