Effect of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 on translocation.
1. The effect of elongation factor 2 (EF 2) and of adenosine diphosphate-ribosylated elongation factor 2 (ADP-ribosyl-EF 2) on the shift of endogenous peptidyl-tRNA from the A to the P site of rat liver ribosomes (measured by the peptidyl-puromycin reaction) and on the release of deacylated tRNA (measured by aminoacylation) was investigated. 2. Limiting amounts of EF2, pre-bound or added to ribosomes, catalyse the shift of peptidyl-tRNA in the presence of GPT; when the enzyme is added in substrate amounts GMP-P(CH2)P [guanosine (beta, gamma-methylene)triphosphate] can partially replace GTP. ADP-ribosyl-EF 2 has no effect on the shift of peptidyl-tRNA when present in catalytic amounts, but becomes almost as effective as EF 2 when added in substrate amounts together with GTP; GMP-P(CH2)P cannot replace GTP. 3. The release of deacylated tRNA is induced only by substrate amounts of added EF 2 and also occurs in the absence of guanine nucleotides. In this reaction ADP-ribosyl-EF 2 is only 25% as effective as EF 2 in the absence of added nucleotide, but becomes 60-80% as effective in the presence of GTP or GMP-P(CH2)P. 4. The results obtained on protein-synthesizing systems are consistent with the hypothesis that ADP-ribosyl-EF 2 can operate a single round of translocation followed by binding of aminoacyl-tRNA and peptide-bond formation. 5. From the data obtained with the native enzyme it is concluded that the two moments of translocation require different conditions of interaction of EF 2 with ribosomes; it is suggested that the shift of peptidyl-tRNA is catalysed by EF 2 pre-bound to ribosomes, and that the release of tRNA is induced by a second molecule of interacting EF 2. The hydrolysis of GTP would be required for the release of pre-bound EF 2 from ribosomes. 5. The inhibition of the utilization of limiting amounts of EF 2 on ADP-ribosylation is very likely the consequence of a concomitant decrease in the rate of association and dissociation of the enzyme from ribosomes.
D000247:Adenosine Diphosphate Sugars; D000818:Animals; D004167:Diphtheria Toxin / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism; D009243:NAD / Q000494:pharmacology; D009702:Nucleoside Diphosphate Sugars; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects; D010445:Peptide Elongation Factors; D011691:Puromycin / Q000494:pharmacology; D012343:RNA, Transfer / Q000378:metabolism; D051381:Rats; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*
D000247:Adenosine Diphosphate Sugars; D004167:Diphtheria Toxin; D009702:Nucleoside Diphosphate Sugars; D010445:Peptide Elongation Factors; D012269:Ribosomal Proteins; D009243:NAD; D011691:Puromycin; D006160:Guanosine Triphosphate; D012343:RNA, Transfer