The Formation of Guanosine-Nucleotide . Elongation-Factor-G . Ribosome Complexes on Free 70-S Ribosomes, 50-S Subunits, and Polysomes. A Comparative Study

Abstract

The ability of free 70-S ribosomes, 50-S subunits and purified, endogenous Escherichia coli polysomes to form complexes with elongation factor G (EF-G) and different concentrations of guanosine nucleotides has been examined. GTP (plus fusidic acid) has been used with all these types of ribosomal particles and GDP (plus the antibiotic), guanyl-5′-yl methylenediphosphonate (Guo-5′-P2-CH2-P) and guanyl-5′-yl imidodiphosphate (Guo-5′-P2-NH-P) with free 70-S ribosomes. In every case the data, obtained with the nitrocellulose membrane filtration technique and under saturating concentrations of EF-G and fusidic acid (when present), fell on one straight line in Scatchard plots, thus indicating that the sites involved in guanosine nucleotide binding are homogeneous and lack cooperative interactions. With 70-S ribosomes between 0.6 and 0.75 molecule of nucleotide were bound per ribosome at saturating concentrations of guanosine nucleotide (3–10 μM). Assuming that about one-third of ribosomes in the preparations were inactive, this result suggests that with each of the four guanosine nucleotides one molecule of this ligand was bound per ribosome. The apparent affinity constants Ka were: for GTP = 7.7 × 106 M−1, for GDP = 1.3 × 107 M−1, for Guo-5′-P2-CH2-P= 5.0 × 106 M−1 and for Guo-5′-P2-NH-P= 1.9 × 106 M−1. 50-S subunits, under the conditions used (10 mM Mg2+ and 65 mM NH+4) and at saturating concentrations of GTP, only complexed about 0.35 molecule of nucleotide per subunit and the apparent KGTPa was 1.2 × 106 M−1. Addition of 30-S subunits to the 50-S subunits raised the number of molecules complexed by 50-S particles to 0.5 and increased KGTPa to 5.1 × 106 M−1, suggesting an important role of the 30-S subunit in the configuration of the nucleotide binding site. With purified endogenous polysomes, washed with either 1 M or 0.1 M NH4Cl, only 0.2–0.25 molecule of GTP was bound per ribosome at saturation and KGTPa was 6.7 × 106 M−1. Deacylation of nascent peptidyl chains raised these values to about 0.5 and 9.6 × 106 M−1, respectively. The small fraction of ribosomes that formed the complex in untreated polysomes appeared to be constituted mainly of true polysomal ribosomes, since (a) they possessed peptidyl-tRNA, as judged by the increased stability of their GDP · EF-G · ribosome · fusidic-acid complex in the presence of puromycin, and (b) they sedimented as polysomes. The results thus indicate the existence of a functional heterogeneity in the polysomal preparations.