Testing the classical two-tRNA-site model for the ribosomal elongation cycle.

An experimental system where the elongation of a polypeptide (polyphenylalanine) is performed stepwise and synchronously by purified Escherichia coli ribosome in a matrix-coupled poly (U) column is proposed for testing the number of non-overlapping tRNA binding sites on the elongating ribosome. If phenylalanyl[3H]tRNA is introduced into the column and bound with the ribosomes at the beginning of a given elongation cycle, deacylated [3H]tRNA is shown to be released from the ribosomes and comes out from the column at the translocation step of the next elongation cycle. The result obtained is fully predicted by the classical two-tRNA-site model and contradicts any model involving more than two non-overlapping high-affinity tRNA binding sites in the ribosomal elongation cycle.