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

Abstract

A population of free, native ribosomal 40S subunits, that do not react with 60S subunits to form 80S ribosomes, has been identified in the postmicrosomal fraction of rat liver homogenates. A protein (IF-3) has been purified from high salt (0.88 M KCI) extracts of native 40S subunits by gradient centrifugation and by ammonium sulfate fractionation; it prevents the reassociation of subunits and to a limited extent dissociates ribosomes to subunits. The activity is measured by ultracentrifugation of the reaction products on linear sucrose gradients, or with an assay developed in this laboratory that couples dissociation with the 60S-specific peptidyltransferase reaction; the latter procedure measures the amount of 60S subunits released from ribosomes or remaining in incubations in the presence of IF-3. Dissociation factor activity is recovered from most of the particles that are resolved by zonal centrifugation of the total "native subunits" obtained from the postmicrosomal fraction; the highest concentration of IF-3, however, appears to be associated with native 40S subunits. The purified dissociation factor IF-3 is composed of about ten polypeptides and the molecular weight is estimated to be between 500 000 and 700 000, on the basis of glycerol and cesium chloride gradient centrifugation. When purified 40S subunits react with IF-3 or when 80S ribosomes are dissociated by IF-3, a product is formed which is dependent on the concentration of the protein factor and has the characteristics of a 40SIF-3 complex; centrifugation of the complex on sucrose and cesium chloride gradients suggests that the complex consists of 1 equiv of each of the two components. Although dissociation factor IF-3 appears to react in a specific manner with free or ribosome-associated 40S subunits, the reaction with subunits differs in several respects from that with ribosomes. The dissociation factor also appears to interact with 60S subunits but multiple complexes are formed, some with more than 1 IF-3 equiv per 60S particle. The IF-3 converts 40S dimers (55S particles) to the 40S-IF-3 complex and dissociates free, native 80S particles present in the postmicrosomal fraction, but it does not affect polysome-associated ribosomes engaged in protein synthesis.

Molecular cloning and functional expression of a human cDNA encoding translation initiation factor 6

“…Two protein factors, eukaryotic translation initiation factor 3 (eIF3) and eIF6, have been implicated in this process (1)(2)(3)(4)(5). It has been reported that eIF3, a multisubunit protein complex of M r Ͼ 500,000, binds to the 40S ribosomal subunit and prevents its association with the 60S ribosomal subunit to form 80S ribosomes (8)(9)(10)(11). In contrast to eIF3, eIF6, a monomeric protein of about 25 kDa, has been shown to bind to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit (12)(13)(14)(15).…”

Interaction of a limited set of proteins with different mRNAs and protection of 5′-caps against pyrophosphatase digestion in initiation complexes

“…Bergmann, J. Gordon and D. Baltimore). It is known that the majority of native 40S subunits contain eIF-3 (33,34), and the same factor binds specifically to 40S subunits in vitro (29,30). Consequently, it is tempting to suggest that in vivo a pre-initiation complex between mRNA and the 24,000 dalton capbinding polypeptide is directed to eIF-3-containing 40S ribosomal subunits at least partly by an interaction between the eIF-3 and the cap binding protein.…”

Ultraviolet-crosslinking reveals specific affinity of eukaryotic initiation factors for Semliki Forest virus mRNA

“…elF-3 is a large complex of ten different subunits ranging from 110 kDa to 34 kDa (Fig. 1, lane C) [38][39][40][41]. The results of the crosslinking experiment (lane D), shows that only three of them are in direct contact with the SFV mRNA molecule.…”