Ribosomes terminated in vitro are in a tight association with non-phosphorylated elongation factor 2 (eEF-2) and GDP

A proportion of the ribosome population in the eukaryotic cell is present in the form of single 80-S ribosomes. These are not involved in translation and are tightly associated with eukaryotic elongation factor 2 (eEF-2). The factor dissociates from ribosomes when it is ADP-ribosylated. Attempts at reconstitution of such complexes from ribosomal subunits and eEF-2 were not successful. We have shown that monomeric ribosomes in a tight complex with eEF-2 can be obtained in vitro as terminated ribosomes in a reconstituted translation system containing isolated polyribosomes, elongation factors and pH5 enzymes (all from rabbit reticulocytes). Incubation of the system with radioactive GTP demonstrated that terminated ribosomes contain GDP. ADP-ribosylation of eEF-2 bound to terminated ribosomes by diphtheria toxin leads to dissociation of both eEF-2 and GDP to the same extent. Thus the presence of GDP in terminated ribosomes is eEF-2 dependent. Ribosomes terminated in vitro as well as native single ribosomes contain the non-phosphorylated form of eEF-2. We assume that tight association of terminated ribosomes with the non-phosphorylated form of eEF-2 excludes both the ribosome and active eEF-2 from the translational cycle and thus, maintains the optimal proportion of translating ribosomes and free eEF-2 in the cell.