Regulation of polysome aggregation in a cell-free system through ammo acid supply

Abstract

Rat-liver polysemes were incubated with an energy-generating system and with activating and transfer enzyme preparations depleted of free amino acids. When no amino acids were added to the reaction mixture, this system incorporated [ 14C]leucine to a limited extent, and attained a plateau within 20 minutes. In the presence of a complete mixture of amino acids, incorporation was more extensive, and continued at a diminishing rate up to 60 minutes. Under both conditions of amino acid supply, extensive and rapid breakdown of polysomes to monosomes and other oligosomes occurred. However, in the presence of added amino acids there was a greater accumulation of free ribosomes, as shown by reaction with polyuridylic acid. It is concluded that the system finally ceases to incorporate in the presence of an adequate supply of amino acids due to the lack of some chain-initiating factor required for the interaction of free ribosomes with messenger RNA. When the system without added amino acids ceased to incorporate the [ 14C]amino acid, it could be restarted by adding a complete mixture of amino acids, but not by adding a mixture lacking any one of the following: arginine, histidine, cysteine, lysine, methionine, phenylalanine, threonine, tryptophan or valine. Polysome aggregates were restored simultaneoulsy with amino acid incorporation; this was accompanied by a reduction in monosomes and other oligosomes with preferential removal of free ribosomes. The resynthesis of polysomes did not occur when mixtures lacking any one of the above named amino acids were added to the incubation. Resynthesis of polysomes could also be prevented by adding ribonuclease (2 μg/ml.), cycloheximide (1 mg/ml.), or puromycin (200 μg/ml.) to the incubation mixture simultaneously with the addition of the amino acids. It is concluded that amino acid supply is one factor regulating attachment of free ribosomes to messenger RMA, and that conditions compatible with peptide synthesis must be provided in order to obtain polysomes.