A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling

Abstract

Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein–EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here.

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

“…Taken together these studies implicate, in agreement with recent studies [14], the possibility of a conserved role of unfolded protein conformation, irrespective of the identity of the protein, in influencing ribosome subunit dissociation. The unfolded protein is expected to be present at substoichiometric levels with respect to the ribosome under physiological conditions, conditions under which negligible 70S dissociation was observed in this study.…”

“…While the binding of tRNA to the A site might hinder access of uBCAII to crucial interaction sites residing in the A loop, the tRNA bound to the P site might interfere in the participation of nucleotides in the P loop whose mutation abolishes chaperoning activity of this RNA domain [33,34]. A recent study has also shown that while RRF, EFG, and GTP are capable of dissociating a tRNA-bound ribosome, binding of tRNA significantly suppresses unfolded-protein-mediated subunit dissociation [14]. In addition, our studies also show that ribosome subunit dissociation mediated by unfolded proteins is similarly inhibited at higher spermidine concentrations (in the presence of 7.5 mM Mg 2+ ; Fig.…”

“…It should be noted that both tRNA and ribosome recycling factor , which makes extensive contacts with the H69 of the large subunit rRNA, inhibit unfolded‐protein‐mediated subunit dissociation (Fig. E and ). In these studies, the ribosome was bound to the antibiotics paromomycin and neomycin under conditions stated in the literature (Materials and methods), and light scattering studies were performed in the presence of uBCAII.…”

“…E). Indeed, a fluorescence anisotropy study reported recently has also shown direct interaction between the unfolded protein and the 70S ribosome . The effect of paromomycin binding on stable association between the 50S subunit and the protein (when present at 1 : 5 ratio) was also studied using dot blot analysis (Materials and methods).…”

The kinetic mechanism of bacterial ribosome recycling

“…For example, as mentioned above, ribosome recycling from a polysome might proceed differently than from a monosome. It has also been suggested that incompletely folded full-length nascent protein newly released from a tRNA-bound ribosome might directly impact recycling ( 60 ). Clearly, further experiments will be required to resolve these points.…”