The exchange of ribosomal subunits during the release of growing polypeptide chains by puromycin has been investigated in a bacterial cell-free system engaged in protein synthesis. The addition of sperinidine, used as a stabilizing agent of 70S monomers, caused a strong inhibition of the subunit exchange. This result led us to conclude that upon premature release of unfinished protein chains by the antibiotic, the ribosomes fall off mRNA as 70S particles. This behavior is different from that occurring during physiological termination of translation, where the ribosomes detach in a dissociated form. Some implications of the postulated mechanism are also discussed.Puromycin is a well-known inhibitor of protein synthesis in vivo as well as in cell-free systems. Its structure, analogous to aminoacyl-tRNA, leads to the premature release of unfinished polypeptide chains as polypeptidyl-puromycin derivatives (1-4).However, the fate of ribosomes upon release of growing protein chains by puromycin is still not well understood. Schlessinger et al. (5) reported that ribosomes engaged in polyphenylalanine synthesis were dissociated into subunits after treatment with puromycin, because the free 30S-50S couples were unstable. More recently Kohler et al. (6) could not confirm these results; they found that 708 particles bearing polyphenylalanyl-tRNA remained intact when peptide chains were liberated by the antibiotic.Both experiments lead to opposite conclusions, but neither of them can be used as a good model of the puromycin reaction at the polysomal level, which constitutes a more physiological system.

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A cell-free system able to elongate and terminate nascent peptide chains has been prepared with polyribosomes and supernatant fraction from Escherichia coli Dlo. When labeled 30-5 subunits were added in several conditions and at different moments, the exchange of radioactivity between 70-5 and 30-5 particles could be measured during and after termination of protein synthesis. The subunit exchange taking place during completion of polypeptide synthesis was not m o a e d by the presence of spermidine, used to stabilize 70-5 ribosomes. This result led to the conclusion that upon termination and release of polypeptide chains, ribosomes dissociate and detach from polysomes as 30-5 and 50-5 subparticles. The "hybrid" 70-5 monomer formed after the exchange with added 30-5 subunits, behaves as a run-off ribosome.When peptide chains are released upon completion of translation, polysomal ribosomes detach from mRNA and have to go through several steps in order to reinitiate another round of protein synthesis. It is now widely accepted that when polypeptide chain synthesis is allowed to terminate under certain experimental conditions, such as carbon-source limitation [l, 21, amino-acid starvation [2,3], met-tRNA exhaustion [4,5] or slow cooling of growing cultures [6], 70-5 ribosomal particles (run-off ribosomes) and not subunits accumulate. This fact suggested the idea that termination implies the ribosomal detachment of their mRNA as 70-5 particles. However, studies in vitro on the exchange of subunits between the ribosomes engaged in translation and the pool of free subparticles have been interpreted as an indication that ribosomes dissociate upon termination to allow the release of 30-5 and 50-S particles from mRNA [7,8].More recently it has been shown in several laboratories that run-off ribosomes are also able to exchange their subparticles with the pool of free subunits [g, 101. These results led to the conclusion that the exchange of ribosomal subunits could take place during or after polypeptide chain termination 31.[9,10]; therefore it is still a matter of discussion whether dissociation is simultaneous with termination of protein synthesis or, on the contrary, the 70-5 ribosomes are released from polysomes and the exchange reflects the equilibrium between runoff 70-5 monomers and ribosomal subparticles. I n an attempt to distinguish between these two possibilities, we have studied the exchange of subunits during and after translation in a cell-free system with endogenous messenger and radioactive 30-S particles, including spermidine in certain conditions. This polyamine proved to be a very useful tool because it behaves as a stabilizer of the monomers already liberated from polysomes [ll]. MATERIALS AND METHODSEscherichia coli D,, was used in all the experiments. Growth conditions, harvesting and lysis procedures were reported previously [6,11].Spermidine phosphate was purchased from Mann and pancreatic ribonuclease type I-A from Sigma. I*C-labeled amino acids (reconstituted protein hydrolysate) were obtained fr...

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