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

<div><p>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 <i>in vivo</i> and in in vitro translation with <i>E</i>.<i>coli</i> 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.</p></div

Dissociation of factors pre-bound to 70S, analyzed using fluorescence light scattering (at 20°C) and 5–20% sucrose density gradient centrifugation.

<p><b>(A)</b> Percent (%) dissociations of 70S by unfolded bovine carbonic anhydrase (UNP) are plotted as a function of time from (70S-EFG-GMPPNP-fusidic acid) complex (■); (70S-EFG-GTP-fusidic acid) complex (●) and (70S-RRF) complex (▲). <b>(B)</b> Light scattering measurements after sequential addition of factor(s) to the 70S and 70S-translation factor complexes: [A] 70S pre-bound to EFG-GMPPNP (0 min) → addition of UNP (at 1 min.); [B] 70S pre-bound to EFG-GMPPNP (0 min) → addition of RRF at 1 min → addition of UNP at 8 minute; [C] Only 70S (0 min.) → addition of RRF at 1 min. → addition of UNP at 8 minute; [D] 70S pre-bound to EFG-GTP (0 min.) → addition of RRF at 1 min. → addition of UNP at 8 minute.</p

Filter binding of tRNA^Glu to 70S.

<p><b>(A)</b> Percent (%) 70S (<i>E</i>.<i>coli</i> wild type) bound to the [α-³²P] UTP labeled tRNA are plotted in Y-axis against tRNA: 70S molar ratio. <b>(B)</b> After binding 70S to [α-³²P] UTP labeled tRNA at 25mM Mg²⁺, reaction mixture was diluted to7mM Mg²⁺ in the subunit dissociation buffer. Bar diagrams show percent (%) 70S bound by [α-³²P] UTP labeled tRNA before (bar 1) and after (bar 2) dilution. Error bars (s.d.) are propagated from 3 independent experiments for each of the bars.</p

CD spectra of native bovine carbonic anhydrase (BCA), denatured BCA, folding intermediate of BCA and refolded BCA (by the PTC-RNA).

<p>Refolding was done following previous protocol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170333#pone.0170333.ref019" target="_blank">19</a>], where guanidine denatured BCA was diluted 100 times in the buffer containing 50mM Tris (pH:7.5), 10mM Magnesium acetate and 100mM NaCl, at 25°C temperature, in presence of 200nM of PTC-RNA. To isolate the folding intermediate, denatured BCA was snap chilled after 2 hours of denaturation reaction and aliquoted out at requisite concentration of denatured protein into the above buffer immediately prior to CD measurement.</p

Dissociation of ribosomal subunits analyzed using fluorescence light scattering (at 20°C) and 5–20% sucrose density gradient centrifugation.

<p><b>(A)</b>Percent (%) dissociations of 70S are plotted as a function of time by: unfolded bovine carbonic anhydrase (UNP) (■); RRF+EFG-GTP+IF3 (●); decapeptide VGDANPALQK (▲); Native bovine carbonic anhydrase (▼). <b>(B)</b>Percent (%) dissociations of 70S by: RRF+EFG-GTP+IF3 (■); UNP+RRF+EFG-GTP+IF3 (●); UNP+EFG-GTP (▲); UNP+RRF (▼) are plotted against time. <b>(C)</b>Percent (%) dissociations of 70S by: UNP+EFG-GMPPNP (■); EFG-GMPPNP (●); EFG-GTP (▲) are plotted against time. <b>(D)</b>Dissociation rate constants (<i>k;</i> s^-1) are derived from the single exponential fits of the respective graphs and plotted as bar graphs against the corresponding combination of factors indicated in the figure. Error bars (s.d.) are propagated from three independent experiments for each combination of factors. <b>(E)</b> P-values for the dissociation rate constants (<i>k</i>) are calculated from three independent experiments for the respective combination of factors as indicated in Fig D and plotted here. Results showing statistical significance at <i>p</i>< 0.05. <b>(F)</b> Sucrose density gradient centrifugation showing dissociation of 70S by: unfolded protein (■); deca-peptide VGDANPALQK (●). The profile of 70S, 50S, 30S (▲) ran in a parallel sucrose gradient; and only untreated 70S (▼) ran in another gradient in parallel, are shown. <b>(G)</b> Sucrose density gradient centrifugation showing dissociation of 70S by: the combinations of RRF, EFG-GTP and IF3 (■);UNP, RRF, EFGGTP and IF3 (●); UNP and EFG-GTP (▲);UNP and RRF(▼).</p

<i>E</i>. <i>coli</i> ribosomal RNA (rRNA) structure derived from PDB structure 3J7Z.

<p><b>(A)</b> Domain V of rRNA is shown in cyan; two r-proteins L4 and L22 are marked that forms narrowest constriction at the peptide exit tunnel; CCA-end of the P-site tRNA is colored yellow; rRNA nucleotides that release unfolded protein slowly are marked red (based on our previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170333#pone.0170333.ref012" target="_blank">12</a>]); rRNA helices that constitute important bridges B2a/B2b, B3 –that join 50S to 30S are marked green (RRF interacts with this region to dissociate 70S). <b>(B)</b> Close-up view showing rRNA nucleotides numbering that release unfolded protein slowly (red) are in close proximity to the rRNA helices constituting B2a/B2b, B3 bridges (green); CCA-end of tRNA is marked yellow.</p

Affinity of ribosome and chaperones for unfolded proteins.

<p>Affinity of ribosome and chaperones for unfolded proteins.</p

Dissociation of ribosomal subunits analyzed using fluorescence light scattering (at 20°C) and 5–20% sucrose density gradient centrifugation.

<p><b>(A)</b> Percent (%) dissociations of (70S-tRNA) complex are plotted as a function of time by: unfolded bovine carbonic anhydrase (UNP) (●); RRF+EFG-GTP+IF3 (■) and RRF+EFG-GMPPPNP+IF3 (▼). <b>(B)</b>Percent (%) dissociations of (70S-tRNA) complex by: UNP+RRF+EFG-GTP (■); UNP+EFG-GTP (●); UNP+EFG-GMPPPNP (▲); and EFG-GTP (★). <b>(C)</b> Dissociation rate constants (<i>k;</i> s^-1) are derived from the single exponential fits of the respective graphs and plotted as bar graphs against the corresponding combination of factors indicated in the figure. Error bars (s.d.) are propagated from three independent experiments for each combination of factors. <b>(D)</b> P-values for the dissociation rate constants (<i>k</i>) are calculated from three independent experiments for the respective combination of factors as indicated in Fig C and plotted here. Results showing statistical significance at <i>p</i>< 0.05. <b>(E)</b> Sucrose density gradient centrifugation showing dissociation of (70S-tRNA) complex by: unfolded bovine carbonic anhydrase (UNP) (■) and RRF+EFG-GTP+IF3 (●).</p