A procedure for isolation of pure complexes of acylated and unacylated tRNA(Phe) with ribosomes from Escherichia coli.

A simple and rapid procedure for the isolation of pure specific complexes of poly(U).ribosome with acylated and/or unacylated tRNA(Phe) is described. The method is based on the use of conditions that favor the formation of polyribosomes. The polyribosomes are separated from ribosomal subunits and monoribosomes by gel filtration on Sepharose 4B at low Mg2+ concentrations that cause the selective dissociation of tRNA-vacant ribosomes from polyribosomes. The procedure allows the isolation in about one hour of various ribosome.poly(U).tRNA complexes completely free of ribosomal particles unable to bind tRNA. A minor fraction of the purified ribosomal complexes is formed by particles devoid of peptidyltransferase activity. Attempts to eliminate this fraction of inactive ribosomes and to obtain ribosomal complexes fully active in polypeptide synthesis were unsuccessful.