Factor-Promoted Dissociation of Free Ribosomes in a Rabbit Reticulocyte Lysate System: Inhibition and Requirement for an Energy Source

Abstract

A factor that promoted the dissociation of ribosomes into ribosomal subunits in a complete globin-synthesizing system was isolated from rabbit reticulocyte ribosomes. The factor stimulated both globin synthesis and the association of the small ribosomal subunit with polyribosomes. The dissociation of free ribosomes by the factor could be followed as an independent reaction in the presence of a high concentration of cycloheximide, which inhibited translation and the resulting accumulation of runoff subunits. This reaction required an energy source and was inhibited by P-(5'-guanylyl)-methylenebisphosphonate. These data suggest that energy generated by hydrolysis of GTP may be a requirement for the dissociation reaction. Aurintricarboxylic acid inhibited the factor-mediated dissociation of free ribosomes but not the factor-mediated preservation of subunits formed on peptide-chain termination.In mammalian cells or cell preparations engaged in protein synthesis, the ribosomes of polyribosomes dissociate into subunits after completion of translation. These subunits undergo exchange with a pool of ribosomal subunits (1-5). Single ribosomes, however, do not readily enter polyribosomes nor do their components exchange with the pool of ribosomal subunits (1-6). When initiation of protein synthesis is decreased, single ribosomes accumulate at the expense of polyribosomes, but essentially a constant amount of subunits is maintained. This change in the ribosome cycle is reversible; single ribosomes return to the polyribosome component and again engage in protein synthesis (7-9). These observations indicate the existence of a mechanism through which free single ribosomes dissociate into subunits which then function in the initiation of peptide-chains.We report here that a fraction that contains an initiation factor (IF) from rabbit reticulocyte ribosomes has dissociation factor (DF) activity for these ribosomes in a complete protein-synthesizing system. This factor also promotes the engagement of the dissociated subunits in globin synthesis. Under conditions of active protein synthesis, it is not possible to discriminate between a role of DF in preservation of subunits released from polyribosomes after peptide chain termination and active dissociation of the free ribosome pool, which is formed by combination of such subunits. Differential inhibition of each of the above reactions is described, and an energy source (or GTP) requirement for dissociation of free ribosomes is established. This factor is shown to participate Abbreviations: DF, ribosomal dissociation factor; IF, peptide chain initiation factor; GDP-CH2P, P-(5'-guanylyl)methylenebisphosphonate. 787 both in the dissociation of free ribosomes and in the preservation of subunits formed after peptide chain termination. METHODS Preparation of a Fraction with

Studies on native ribosomal subunits from rat liver. Purification and characterization of a ribosome dissociation factor

“…The rat liver factor purified in this laboratory appears to be active at more physiological and higher (1.6 to 5.6 mM) magnesium ion concentrations than some reported with factors from reticulocyte (Lubsen and Davis, 1972;Merrick et al, 1973;Mizuno and Rabinowitz, 1973), ascites cells (Ayuso-Parilla et al, 1973), or rat liver (Lawford et al, 1971). It should also be noted that, although a requirement for nucleotides (ATP or GTP) has been reported with some preparations of dissociation factor (Mizuno and Rabinowitz, 1973;Favelukes et al, 1973;Chen et al, 1975), the reactions described here do not require nucleotides.…”

“…al., 1968, 1969); this dissociation activity is expressed by purified IF-3 from prokaryotes (Sabol and Ochoa, 1971). Ribosome dissociation activity has also been reported in the ribosomal "wash" fraction or partially purified preparations of yeast (Petre, 1970), reticulocytes (Lubsen and Davis, 1972;Merrick et al, 1973;Mizuno and Rabinowitz, 1973;Favelukes et al, 1973; Chen et al, 1974), and ascites cells (Nakaya et al, 1973), and in extracts obtained from native subunits of Ehrlich ascites cells (Ayuso-Parilla et al., 1973), reticulocytes (Lubsen and Davis, 1974), a rat liver fraction rich in native subunits (Lawford et al, 1971), and rat liver native 40S subunits (Sadnik et al, 1975).…”