Interaction of rat liver ribosomal subunits with homologous Met-tRNAs

Abstract

Rat liver 40 S ribosomal subunits, in the presence of magnesium ions, bind homologous, resolved Met-tRNAs in the absence of added exogenous proteins. The interaction of the aminoacyl-tRNAs with the particle is dependent on the concentration of magnesium ions in the incubation. At various Mg 2+ concentrations examined, binding of the putative initiator Met-tRNA i to 40 S subunits is greater than that observed with Met-tRNA m. Also, binding of Met-tRNA i to 40 S subunits is greater than that obtained with 40 S plus 60 S particles. The initial rate of formation of the 40 S·Met-tRNA i complex is greater at 25 °C than at 37 or 4 °C; decay of the complex, which is observed after 15 min of incubation, is greater at 37 °C but it is slower if 60 S subunits are added after the complex has been formed. If 60 S subunits are added to the incubation with 40 S subunits at the start of the reaction, binding of Met-tRNA i is inhibited; inhibition is also obtained if elongation (binding) factor EF-1 or stripped tRNAs (particularly tRNA Met) are present in the incubation mixture containing 40 S subunits. Acetyl-Met-tRNA i binds to 40 S·ApUpG complex to the same extent as unacetylated Met-tRNA i and, after addition of 60 S subunits, reacts extensively with puromycin; the addition of elongation (translocation) factor EF-2 and GTP do not affect the extent of the puromycin reaction, suggesting that the acMet-tRNA i is bound to a site on the 40 S subunits which becomes the P site on 80 S ribosomes.