Control of Single Ribosome Formation by an Initiation Factor for Protein Synthesis

Abstract

ABSTRACT30 and 50S ribosomal subunits, released from polysomes upon polypeptide chain termination, possess a high affinity for each other and readily form single ribosomes. Highly purified initiation factor F3(B), acting stoichiometrically, prevents the formation of single ribosomes without promoting their dissociation. These findings are interpreted in terms of a ribosome cycle in which a limiting amount of factor F3(B) controls the number of ribosomes active in protein synthesis, in response to metabolic changes in the cell, by regulating the flow of ribosomal subunits into polysomes or into a sidetrack pool of synthetically inactive single ribosomes. The reported apparent ribosome dissociation activity of F3(B) is explained.In a cell, ribosomes occur in different functional states: ribosomes actively engaged in protein synthesis (present in polysomes), large and small ribosomal subunits, and single ribosomes. Single ribosomes, in contrast to monosomes, do not carry messenger RNA or growing polypeptide chains and are not participating in protein synthesis. Both in proand eukaryotic cells, translating ribosomes frequently undergo exchange of their two subunits, apparently by dissociation after each passage over messenger RNA and reformation from a pool of ribosomal subunits that continuously recycle through polysomes (1-5). By contrast, single ribosomes, at least in mammalian cells, are not in rapid equilibrium with polysomes or ribosomal subunits (6-9).When the over-all rate of protein synthesis decreases, single ribosomes accumulate at the expense of polysomes, while the size of the ribosomal subunit pool remains essentially unchanged (6,8,10,11). The accumulation of single ribosomes was first interpreted as evidence that they are released from polysomes on polypeptide chain termination, and thus are obligatory intermediates of the ribosome cycle intervening between polysomes and the ribosomal subunit pool (11)(12)(13)(14). Accordingly, the dissociation of single ribosomes would be required for protein synthesis to proceed. One of the three known initiation factors for protein synthesis found associated with 30S ribosomal subunits (15), F3(B)* (16-19), also appears to dissociate single ribosomes under defined conditions in vitro (18-21), and was postulated to mediate the dissociation step (20,21).Recently, however, evidence was presented that ribosomes dissociate directly into subunits upon termination of protein synthesis, and that single ribosomes are formed by association of subunits present in the pool (22). Protein synthesis in Escherichia coli extracts diluted sufficiently to prevent the reformation of ribosomes from subunits results in the conversion of polysomes to ribosomal subunits, not single ribosomes, even though single ribosomes do not noticeably dissociate under these conditions. When protein synthesis is allowed to occur in a more concentrated mixture of isotopically heavy and excess light cell extracts, single ribosomes accumulate; those originating from fully heavy polysomes are entirely of ...

Lack of stable initiation factor 3 (IF‐3) binding to dimers of the 30 S ribosomal subunits

“…Recent studies have shown that IF-3 acts as antiassociation factor since the equilibrium between 70 S ribosomes and 50 S and 30 S ribosomal subunits is shifted in favour of the subunits by the binding of IF-3 to free 30 S particles [3][4][5]. The finding that IF-3 does not stably bind to the 70 S monomers is consistent with this scheme [6][7][8].…”

NEURONAL PERIKARYA WITH DISPERSED, SINGLE RIBOSOMES IN THE VISUAL CORTEX OF MACACA MULATTA

“…Perhaps the axoplasmic flow has been temporarily stemmed, perhaps a preceding surge in synthesis has exhausted the supply of substrates, mRNA, or initiation factors. Kaempfer (15) suggested that the balance between a critical initiation factor like IF-3, which is usually in short supply, and the ribosomal subunits could provide a mechanism for the control of the number of active ribosomes and thus of protein synthesis. Whatever the reason, it would appear likely that the suspension of protein synthesis is a phase in the cyclic function of these cells.…”

A Trichodermin‐Resistant Mutant of Saccharomyces cerevisiae with an Abnormal Distribution of Native Ribosomal Subunits

“…However, if an underproduction of 40-S subunits does indeed occur, then the level of free active 40-S anti-association factor (in the cytoplasm) may be higher than normal. Previous studies [27] have demonstrated that frequent subunit exchange takes place iz7 vivo between 80-S free couples and native subunit pools (presumably through exchange of anti-association factor) so it should be possible to generate a normal complement of native 40-S subunits in CLP-8 by increasing the amount of 80-S free couples. Accordingly, intact spheroplasts from both CLP-8 and A224A were incubated for 15 min at 30 'C in the presence of sodium azide (1 mM).…”

“…In growing yeast cells frequent subunit exchange occurs between 80-S free couples and the native ribosomal subunit pool with no evidence of a fraction of 80-S ribosomes unable to dissociate or for accumulation of subunits incapable of recycling [27]. To ascertain if the native 60-s* peak of CLP-8 is functionally homogeneous and capable of such recycling we developed an assay system for protein synthesis in vitro utilizing either high-salt-washed 80-S ribosomes or purified ribosomal subunits, a crude factor preparation and exogenous mRNA, i.e.…”