Reduced puromycin sensitivity of translocated polysomes after the addition of elongation factor 2 and non-hydrolysable GTP analogues.

Treatment of reticulocyte polysomes with elongation factor eEF-2 and GTP led to an increased sensitivity of peptidyl-tRNA for puromycin as a result of the translocation from the ribosomal A-site to the P-site. Upon addition of an excess of the non-hydrolysable GTP analogue, GuoPP[CH2]P, the puromycin sensitivity decreased rapidly. The decrease in sensitivity required high concentrations of eEF-2 with half maximal effect at an eEF-2 concentration of around 1 microM. The data suggest either that peptidyl-tRNA had re-translocated back to the A-site due to the higher affinity of eEF-2 for the pre-translocation than for the post-translocation ribosome, or that the eEF-2-GuoPP[CH2]P complex blocks the peptidyl-transferase activity.