Termination of Polypeptide Synthesis in Bacteria and the Exchange of Ribosomal Subunits

Abstract

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...

Mechanism of Puromycin Action: Fate of Ribosomes after Release of Nascent Protein Chains from Polysomes

“…If, upon treatment with the antibiotic, ribosomes were dissociated in order to detach from mRNA, the polyamine should not inhibit the subunit exchange, unless the resultant subparticles were unable to equilibrate with the pool of subunits. [This possibility was ruled out by the experiments carried out in the absence of puromycin (11).] On the contrary, if detachment occurs as 70S particles, spermidine would stabilize the monomers and the exchange would be blocked.…”

“…However, it has been shown that this hypothesis is not correct, because run-off 70S monomers are also able to undergo subunit exchange through their equilibrium with the pool of free subunits (11,14).…”

“…This comparison led us to the conclusion that upon physiological termination, when completed polypeptides are released, the ribosomal particles detach from mRNA as subunits, which can then reassociate to form 70S monomers (11).…”