Association Factor of Ribosomal Subunits from Bacillus stearothermophilus

Abstract

The isolation of a new factor, which can cause the in vitro association of 30S and 50S ribosomal subunits at low Mg++ concentration, is described. The association factor is eluted together with the dissociation protein when ribosomes of Bacillus stearothermophilus are washed with salt solutions of high concentration. The association activity is heat-stable, whereas dissociation factor is inactivated after 10 min at 80'C. This treatment allows the separation of both factors. Several properties rule out the possibility that uncharged, aminoacyl-, or peptidyl-tRNA are responsible for the association process described in this report. Digestion with trypsin shows that the association factor contains at least two components, one of which is a protein.Bacterial ribosomes can be found as 70S particles, either free or complexed in monosomes and polysomes, and as 30S and 50S subunits (1). Several different conformations of each type of particle may also exist (2-8).Much evidence has accumulated to show that a cycle of ribosome dissociation and association of 30S and 50S subunits to 70S monosomes occurs during the successive rounds of protein synthesis (1, 9-12). The conditions that favor dissociation and association have been thoroughly studied. 70S ribosomes tend to dissociate with increasing temperature (13), in the presence of sulfhydryl reagents (14), or when the ratio of monovalent-to divalent-ion concentrations is high (15,16).A protein factor that causes the dissociation of 70S particles into 30S and 50S subunits has been recently obtained from different bacteria (17)(18)(19)(20). This factor (DF), necessary for cell survival, presumably dissociates ribosomes in vivo (12).Ribosomal subunits tend to reassociate at high Mg++ concentration (21), in the presence of polyamines (22), or when the subparticles are bound to peptidyl-tRNA or aminoacyl-tRNA (21).In a recent study on the effect of several antibiotics, we observed that neomycin was also able to reassociate subunits (23); this associating capacity of the antibiotic was markedly enhanced in the presence of a heated ribosomal wash fraction from Bacillus stearothermophilus. The possibility arose that an association factor (AF) could be extracted, along with initiation and dissociation factors, from ribosomes of the thermophilic bacteria by salt solutions of high concentration.In this report we will describe the isolation and some properties of a factor that can produce the in vitro association of ribosomal subunits to form a monomer at a relatively low Mg++ concentration, similar to the physiological level.Several attempts to detect the reversibility of ribosomal dissociation as mediated by DF have failed, except when the concentration of Mg++ was increased (1, 17). MATERIALS AND METHODS Escherichia coli Dio and B. stearothermophilus 1503-4Rwere grown as indicated (18).Trypsin (3-times crystallized) was purchased from Worthington; soybean trypsin-inhibitor (Worthington) was a generous gift from Dr. E. Cabib. RNase-free sucrose was obtained from Mann and tRN...

Association of ribosomal subunits: Studies on the binding of polyamines to 30S and 50S particles

“…When the association of subunits was performed in a single step, the reaction was carried out as previously described [9], but using spermidine instead of AF. promoted in a single step reaction by different concentrations of spermidine.…”

“…After incubation for 5 min at 0°C the samples were analyzed by centrifugation in 4.6 ml of 5-20% linear sucrose gradients as previously reported [ 111. Percentages of association were calculated as detailed earlier [9].…”

“…Our studies on the association of ribosomal subunits promoted by an association factor (AF) extracted from ribosomes of Bacillus stearothermophilus [9], have indicated that AF could bind functionally to 30s but not to SOS particles [lo]. These findings and the fact that polyamines seem to be the active components of AF [ 1 I] led us to investigate the correlation between the binding of the above mentioned organic cations to each one of the ribosomal subparticles and the association reaction.…”

Association and dissociation of Neurospora crassa cytoplasmic ribosomes

“…The dissociation assay was performed in the following way: about 0.6 A260 units of 77S ribosomes were incubated at 300C for 10 min with DF in a total volume of 0.2 ml of buffer II. The incubation mixture was then analyzed by sucrose gradient centrifugation as described previously (2,3).…”

“…The mixtures were then analyzed as described above by using sucrose gradients made up in 10 mM Tris-hydrochloride buffer (pH 7.9), 1 mM MgCl2, and 100 mM KCI. Dissociation and association were calculated as described elsewhere (2,3).…”