Binding interactions between radiolabeled Escherichia coli elongation factor G and the ribosome.
We have prepared homogeneous radiolabeled Escherichia coli Elongation Factor G (EF-G) and examined its interactions with the ribosome. In agreement with earlier indirect observations we found that in the presence of high concentrations of fusidic acid approximately equimolar amounts of [3H]EF-G and [alpha-32P]GDP are stably bound to the ribosome. In the absence of fusidic acid, we observed a previously undescribed nucleotide-independent binding interaction between EF-G and the ribosome. This binding is detectable by rapid elution on small gel columns but is not apparent when reactions are analyzed by sucrose density gradient sedimentation. With the exception of the fact that the nucleotide-independent binding of EF-G to ribosome is apparently unaffected even by high concentrations of fusidic acid, it shares many properties in common with that binding which occurs in the presence of GDP. Nucleotide-independent binding requires magnesium ion (10 to 20 mM ) and does not require a monovalent cation but is strongly inhibited by even moderate concentrations of NH4Cl. This binding requires the presence on the ribosome of Protein L7/L12 and is inhibited by the antibiotic thiostrepton. Although we were unable to examine the binary ribosome.EF-G complex by equilibrium means, the observed stoichiometry under the conditions we employed did not exceed 0.2 mol of EF-G/mole of ribosome. Nonequilibrium measurements revealed that one-half of the EF-G was bound at a ribosome concentration of about 50 muM.
D001665:Binding Sites; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000378:metabolism*; D005672:Fusidic Acid / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D010441:Peptide Chain Elongation, Translational; D010445:Peptide Elongation Factors; D010761:Phosphorus Radioisotopes; D011485:Protein Binding; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*
D010445:Peptide Elongation Factors; D010761:Phosphorus Radioisotopes; D005672:Fusidic Acid; D008274:Magnesium