The mechanism of the protein-synthesis elongation cycle in eukaryotes. Effect of ricin on the ribosomal interaction with elongation factors.
The functional significance of the post-translocation interaction of eukaryotic ribosomes with EF-2 was studied using the translational inhibitor ricin. Ribosomes treated with ricin showed a decreased rate of elongation accompanied by altered proportions of the different ribosomal phases of the elongation cycle. The content of ribosome-bound EF-2 was diminished by approximately 65% while that of EF-1 was unaffected. The markedly reduced content of EF-2 was caused by an inability of the ricin-treated ribosomes to form high-affinity pre-translocation complexes with EF-2. However, the ribosomes were still able to interact with EF-2 in the form of a low-affinity post-translocation complex. Ricin-treated ribosomes showed an altered ability to stimulate the GTP hydrolysis catalysed by either EF-1 or EF-2. The EF-1-catalysed hydrolysis was reduced by approximately 70%, resulting in a decreased turnover of the quaternary EF-1 X GTP X aminoacyl-tRNA X ribosome complex. In contrast, the EF-2-catalysed hydrolysis was increased by more than 400%, despite the lack of pre-translocation complex formation. The effect was not restricted to empty reconstituted ribosomes since gently salt-washed polysomes also showed an increased rate of GTP hydrolysis. The results indicate that the EF-1- and EF-2-dependent hydrolysis of GTP was activated by a common center on the ribosome that was specifically adapted for promoting the GTP hydrolysis of either EF-1 or EF-2. Furthermore, the results suggest that the GTP hydrolysis catalysed by EF-2 occurred in the low-affinity post-translocation complex.
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000502:physiology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006868:Hydrolysis; D010441:Peptide Chain Elongation, Translational; D020648:Peptide Elongation Factor 1; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism* / Q000502:physiology; D014176:Protein Biosynthesis; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000201:enzymology / Q000378:metabolism*; D012276:Ricin / Q000494:pharmacology*
D020648:Peptide Elongation Factor 1; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D006160:Guanosine Triphosphate; D012276:Ricin; D020963:GTP Phosphohydrolase-Linked Elongation Factors