Binding of tRNA to Escherichia coli Ribosomes as Measured by Velocity Sedimentation

Abstract

The binding of initiator and elongator tRNA to E. coli 70S ribosomes and its subunits was studied by velocity sedimentation in the analytical ultracentrifuge. This technique shows the advantage over the previously used methods (adsorption of the complexes to nitrocellulose filters or fluorescence titrations) in that no kinetic effects obscure the equilibrium data and that none of the components has to be chemically modified. The concentrations of the macromolecular compounds are kept constant and the binding equilibria are shifted by varying the Mg2+ concentration in a range which is accessible to experimental analysis. Free 30S ribosomes bind no tRNA, whereas 1 tRNA molecule is bound to 50S ribosomal subunits. In the presence of the cognate codon 1 tRNA can be associated with the small subunit. Free, programmed or misprogrammed 70S ribosomes bind exactly 2 elongator tRNA species. Only the initiator tRNA does discriminate significantly between the 2 ribosomal sites when bound to a ribosome.cntdot.AUG complex.