The Binding of Purified Phe-tRNA and Peptidyl-tRNAPhe to Escherichia coli Ribosomes

Abstract

The binding of purified Gly3-Phe-tRNA to Escherichia coli ribosomes was compared to that of crude Gly3-Phe-tRNA (containing bulk tRNA). Gly3-phe-tRNA from the purifed preparation binds preferentially to the P site (peptidyl site) at both a high (30 mM) and a low (10 mM) magnesium acetate concentration, while peptidyl-tRNA from the crude preparation bound more to A site (aminoacyl site) than to the P site at 30 mM magnesium acetate under the same experimental conditions. The addition of tRNAPhe to the purified Gly3-Phe-tRNA preparation strongly inhibited the binding at 10mM. At 30 mM Mg2+ the addition of the same amount of tRNAPhe caused the Gly3-Phe-tRNA to bind partially to the A site, but did not inhibit the total amount of Gly3-Phe-tRNA which binds to the ribosomes. The binding of purified Phe-tRNA in the presence of T factor and GTP is not inhibited by tetracycline. However, tetracycline strongly inhibited the enzymatic binding of Phe-tRNA when tRNAPhe was added. From the results presented in this paper it is proposed that in the presence of poly(U), Phe-tRNA or peptidyl-tRNAPhe can bind to A sites only of those ribosomes which have their P site occupied by tRNA or one of its derivatives (aminoacyl-tRNA or peptidyl-tRNA).