Unfolded protein exhibits antiassociation activity toward the 50S subunit facilitating 70S ribosome dissociation

Abstract

The ability of the ribosome to assist in the folding of proteins both in vitro and in vivo is well documented. The interaction of an unfolded protein with the peptidyltransferase center of the bacterial large ribosomal subunit is followed by release of the protein in a folding-competent state and rapid dissociation of ribosome into its subunits. Our studies demonstrate that the 50S subunit-associated antiassociation ability of an unfolded protein might contribute significantly to its ability to mediate energy-independent and stable dissociation of the ribosome into its subunits. The stoichiometry of the protein present with respect to the ribosome is an important factor in determining whether the ribosome has a chaperoning effect on protein folding or if the protein acts as a 50S subunit antiassociation factor. Sustained interaction of the protein with the ribosome at higher protein concentrations and the hindrance in the formation of the central intersubunit bridge B2a could underlie the antiassociation activity of unfolded proteins. The ribosome dissociation and antiassociation activity of unfolded proteins could make the ribosome susceptible to cellular ribonucleases, thereby initiating ribosome degradation, which is a well-documented phenomenon under nutrient deprivation conditions.

Hibernating ribosomes exhibit chaperoning activity but can resist unfolded protein‐mediated subunit dissociation

“…Our earlier ex‐vivo studies performed using E . coli cell lysate had demonstrated that, the isolated ribosomal subunits formed due to the unfolded protein‐mediated 70S Ec ‐free dissociation, increased the susceptibility of the ribosome towards degradation by cellular ribonucleases [19]. In the present study, similar assays were performed in which 0.1 µ m 70S Ec was incubated with a 10‐fold stoichiometric excess of HPF and YfiA in the presence of 0.5 µ m uBCAII.…”

“…It might be argued that the inability of the unfolded protein to associate with the HPF or YfiA bound 70S Ec ribosome could have prevented the initiation of ribosome splitting. In our earlier studies, the ribosome‐uBCAII complexes were separated from the unassociated protein by ultrafiltration and detected by western blot analysis using anti‐BCAII antibody [19]. Similar experiments were performed to analyse whether uBCAII and the factors HPF and YfiA could simultaneously bind to the ribosome under our experimental conditions.…”

“…The ability of the cell to respond to the increase in protein unfolding determines its resilience under stress conditions. Since the accumulation of unfolded proteins would increase the possibility of their encounter with the ribosome, two important aspects of ribosome‐unfolded protein interaction that are well documented in literature become especially relevant: (a) the isolated subunits formed due to unfolded protein‐mediated ribosome subunit dissociation [17,18] have increased susceptibility towards degradation by cellular ribonucleases [19] and (b) the ability of the ribosomes from diverse origins to assist in the folding of proteins with a wide range of folds and functions which is widely reported in the literature [20] and references therein]. Such noncanonical chaperoning activity of the translation machine could be effectively inhibited in an actively translating ribosome due to the tRNA positioned at the P‐site [21] thus ensuring that an actively translating ribosome does not engage in chaperoning function.…”

Coagulation Factor XIIIA: biochemical properties underlying physiological function.

“…Experimental investigation of RNA binding to the GST-FXIIIA fusion protein is beyond the scope of the current project, and instead the following speculation may be offered. It is well known that unfolded proteins bind to the 50S subunit of the prokaryotic ribosome, and in a recent report, Pathak et al (119) demonstrated that the presence of an unfolded protein in above stoichiometric amounts, promotes dissociation of ribosome subunits, thereby reducing the fold-assisting function of the ribosome. As suggested above, at the relatively high incubation temperature, the FXIIIA polypeptide is assembled faster than it folds into the native conformation.…”

“…This unfolded FXIIIA may thus have caused the dissociation of E. coli ribosomes. Such dissociated ribosomes are susceptible to degradation by cellular ribonucleases (119). Whether the actual ribosome degradation has or has not taken place in the initial 'IPTG expression' efforts, it is possible that some ribosomal RNA remained bound with the fusion protein on the column despite excessive washing steps.…”