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

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Abstract
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 tighly 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 ribosomes and active eEF‐2 from the translational cycle and thus, maintains the optimal proportion of translating ribosomes and free eEF‐2 in the cell.