Detection of Guanosine‐Nucleotide · Elongation‐Factor‐G Complexes Produced during the Decay of Guanosine‐Nucleotide · Elongation‐Factor‐G · Ribosome Complexes

Abstract

The mechanism of formation and decay of complexes containing guanosine nucleotide, elongation factor G (EF‐G) and the ribosome (with or without fusidic acid) has been examined in a purified Escherichia coli system. With a [14C]GDP · [3H]EF‐G · ribosome · fusidic‐acid complex in the presence of either unlabelled GDP plus EF‐G or the antibiotic thiostrepton, it has been found that [14C]GDP and [3H]EF‐G are simultaneously released, which suggests that they dissociate from the ribosome as a binary complex. In fact, guanosine‐nucleotide · EF‐G complexes, which are too unstable to be isolated, seem to be present in mixtures containing either the [3H]GPD · EF‐G · ribosome · fusidic‐acid complex, the [3H]guanyl‐5′‐yl‐methylene‐diphosphonate · EF‐G · ribosome complex, or the [3H]guanyl‐5′‐yl‐imidodiphosphate · EF‐G · ribosome complex, since ribosomes added to these mixtures, together with a 100‐fold excess of the corresponding unlabelled guanosine nucleotide, specifically bind substantial amounts of radioactive guanosine nucleotide.The binary complexes in these mixtures seem to proceed exclusively from the decay of ternary or quaternary complexes, since the same detection procedure fails to reveal binary complexes in mixtures containing only EF‐G and either [14C]GTP, the labelled GTP analogs or [3H]GDP. Moreover, only very small amounts of [3H]GDP · EF‐G complexes are detected in mixtures where either the EF‐G plus ribosome‐dependent GTP hydrolysis or the interaction of [3H]GDP with EF‐G and ribosomes in the absence of fusidic acid is taking place. The results suggest that EF‐G, after binding to the ribosome in the presence of either GTP or its nonhydrolyzable analogs (but not GDP), undergoes a conformational change and retains guanosine nucleotide more strongly. With GTP, the hydrolysis of this nucleotide would reverse this conformational change, facilitating both the release of GDP · EF‐G from the ribosome and the dissociation of this binary complex. Fusidic acid would interfere with the conformational transitions by preserving the prehydrolytic conformation after GTP hydrolysis and/or by inducing it after formation of the GDP · EF‐G · ribosome complex.