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

Date Published: January 18, 2017

Publisher: Public Library of Science

Author(s): Debasis Das, Dibyendu Samanta, Arpita Bhattacharya, Arunima Basu, Anindita Das, Jaydip Ghosh, Abhijit Chakrabarti, Chanchal Das Gupta, Tzvetanka D. Dinkova.


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.

Partial Text

In bacteria, termination of protein synthesis takes place when Class I release factor recognizes a stop codon on the mRNA. Then the nascent protein is cleaved off from the peptidyl tRNA, generating the post-termination complex consisting of 70S ribosome, the mRNA and the deacylated P-site tRNA. The release factors in E.coli, RF1/RF2 are released by RF3 to allow RRF to independently occupy ribosome in presence of EFG and IF3 [1–5]. They carry on “ribosome recycling”, the “fourth step” of protein synthesis, on the post-termination ribosome to initiate a fresh round of translation. RRF alone has been shown to dissociate the 70S partially in a cryo-EM study [6]. The GTPase activity of EFG plays crucial role along with RRF in dissociation, and IF3 sequesters the freed 30S subunit [7] to stabilize the dissociated state [4]. The gene encoding RRF (frr) is essential for bacteria [8]. Its deletion causes unscheduled translation termination [1]. However no proper eukaryotic homolog of it has been identified yet.

At the end of an authentic translation termination in bacteria, class 1 release factor (RF1/RF2) recognizes stop codon on the mRNA and triggers peptidyl tRNA hydrolysis to free the nascent full-length protein as well as the last P-site tRNA. Then the deacylated tRNA is dissociated from 70S in presence of EFG-GTP by RRF [3–5] or unfolded protein [20]. EFG-GTP to EFG-GDP transformation is associated with a structural transition of 70S—a prerequisite for tRNA dissociation. In a recent cryo-EM report [14] it has been shown that RRF promotes the rotated state [39]of ribosome and destabilizes the H69 and H44, thereby EFG-GTP facilitates RRF reorientation and actively splits the B2a bridge connecting 50S and 30S (Fig 6). Thus a common aspect of EFG function at the molecular level has been reflected in both elongation and ribosome recycling phases of translation. While in another study [12] comparing interaction of nascent unfolded protein and chemically unfolded protein with the rRNA also revealed differential affinity of rRNA nucleotides to the different regions of the unfolded protein(s). Those rRNA nucleotides identified to release slowly the C-terminal region of the protein are located in close proximity to the B2a bridge (Fig 6). Hence splitting of 70S by unfolded protein can be conceived as the regulation of rRNA nucleotides that slowly release the unfolded polypeptides at the end of translation and EFG-GTP functions identically as it does during translation elongation to promote disruption of B2a bridge. Present study indicating the ability of both the RRF and the unfolded protein to dissociate tRNA from the ribosome by interacting along with EFG, also point towards that mechanism. The observation that the structure of C-terminal end of nascent peptide is important in translation termination [40] is also relevant in this regard. Because of the independent role of unfolded protein in tRNA dissociation, it can remain bound post-translationally to the ribosome even after its dissociation into subunits. That is why we get a large population of nascent full length protein bound to the 50S both in vivo and in the translation system with the S30 extract [11, 12]. Moreover, our present study of ribosome recycling with the chemically unfolded protein also point towards a chemically conserved interaction between rRNA and unfolded conformation of protein that has evolved from the RNA world [31].




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