Influence of Guanine Nucleotides and Elongation Factors on Interaction of Release Factors with the Ribosome

Abstract Release of formylmethionine from the reticulocyte ribosomal substrate, f[3H]Met-tRNA·ribosome, is promoted by reticulocyte release factor (RF). The initial rate of this reaction is stimulated by GTP but inhibited by GDPCP. Formation of an RF·UA[3H]A2·ribosome complex is a measure of the binding of reticulocyte RF to the ribosome, and the recovery of this complex is increased by GDPCP and, to a lesser extent, GTP. These studies suggest that GTP is involved in the initial association of RF with the ribosome and that hydrolysis of the γ-phosphate of the guanine nucleotide is required at a subsequent rate-limiting step. The ribosomal-dependent fMet-tRNA hydrolysis and GTPase activities of reticulocyte RF are inhibited when elongation factor (EF)-2 is bound to the respective ribosomal substrate in the presence of fusidic acid and GDP. When EF-G is bound to the f[3H]Met-tRNA·AUG·ribosome substrate with fusidic acid and GDP, the fMet-tRNA hydrolysis activity of Escherichia coli RF-1 and RF-2 is also inhibited. The binding of reticulocyte RF and E. coli RF-1 or RF-2 to their respective ribosomes is prevented when fusidic acid·EF-2/EF-G·GDP·ribosome complexes are used.