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.
Davydova
E K
EK
Institute of Protein Research, Russian Academy of Sciences, Moscow Region.
Malinin
N L
NL
Ovchinnikov
L P
LP
D000818:Animals; D002474:Cell-Free System; D015180:Electrophoresis, Gel, Two-Dimensional; D006153:Guanosine Diphosphate / Q000032:analysis / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000032:analysis; D010443:Peptide Chain Termination, Translational; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011817:Rabbits; D012156:Reticulocytes; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*
D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate