Binding of tRNA to Escherichia coli ribosomes as measured by velocity sedimentation.

We followed the binding of initiator and elongator tRNA to 70-S ribosomes and its subunits 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 30-S ribosomes bind no tRNA, whereas one tRNA molecule is bound to 50-S ribosomal subunits. In the presence of the cognate codon one tRNA can be associated with the small subunit. Free, programmed, or misprogrammed 70-S ribosomes bind exactly two elongator tRNAs. Only the initiator tRNA does discriminate significantly between the two ribosomal sites when bound to a ribosome . A-U-G complex.