Structural and Functional Properties of Ribosomes Crosslinked with Dimethylsuberimidate

To test whether a 30S ribosomal subunit-formylmethionyl-tRNA-mRNA complex is an obligatory intermediate in protein synthesis, 70S ribosomes from Escherichia coli were crosslinked with the bifunctional imidoester, dimethylsuberimidate. Crosslinked ribosomes contained covalently joined 30S and 50S subunits, as judged by their inability to dissociate at low Mg2+concentrations. Treatment of 70S ribosomes with high salt (1 M NH4Cl), either before or after reaction with the crosslinking reagent, produced two different crosslinked ribosomal particles, one of ``60 S'' and the other ``70 S.'' Preliminary evidence indicates that both particles can bind N-acetylphenylalanyl-tRNA at low Mg2+concentrations and are active for polyphenylalanine syntheses. Crosslinked ribosomes were functional when tested with poly(U) as an mRNA in systems requiring initiation factors and N-acetylphenylalanyl-tRNA for activity. Under optimal crosslinking conditions, they retained 80% of the activity of unmodified ribosomes for polyphenylalanine synthesis. Despite the maintenance of these functional capacities, such ribosomes had a sharply reduced ability to bind fMet-tRNA and were completely inactive in protein synthesis with bacteriophage f2 RNA as a messenger. We conclude that 70S ribosomes must dissociate into subunits to initiate protein synthesis with natural mRNAs.