Effects of tRNA on chaperoning ability of non-programmed ribosome.

<p>(<b>A</b>) Dose dependent inhibition of ribosome's chaperoning ability due to binding of total <i>E. coli</i> tRNA. <i>E. coli</i> ribosome was allowed to bind to increasing concentrations of total <i>E. coli</i> tRNA in Buffer-P (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101293#pone.0101293.s003" target="_blank">Table S1</a>). Reactivation of BCAII by the ribosome-tRNA complex was plotted (▪) against increasing total <i>E. coli</i> tRNA (▪) concentrations. The refolding experiments were repeated at least 3 times and the average values were plotted. The graph was fitted by Boltzmann fit. (<b>B</b>) Percent (%) reactivation of BCAII in presence of total <i>E. coli</i> tRNA and Met-tRNA bound to 70S ribosome in Buffer-A and Buffer-P (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101293#pone.0101293.s003" target="_blank">Table S1</a>) are represented. (<b>C</b>) Effects of tetracycline on tRNA bound ribosome assisted refolding; Bar diagram represents a comparison of the BCAII reactivation (%) of; Self (1), 70S ribosome (2), 70S ribosome + tetracycline (3) 70S ribosome +5 µM total <i>E. coli</i> tRNA (4), 70S ribosome + tetracycline +5 µM total <i>E. coli</i> tRNA (5), 70S ribosome +5 µM deacylated Met-tRNA (6) and 70S ribosome + tetracycline +5 µM deacylated Met-tRNA (7). BCAII reactivation in absence of chaperone is marked as ‘Self’ in the figure.</p

Representation of the structure of <i>E. coli</i> ribosome with P-site tRNA.

<p>(<b>A</b>) The 23S rRNA of <i>E. coli</i> large ribosomal subunit (PDB: 2I2V) has been displayed (light orange) here with 3′-CCA end of P-site tRNA (PDB: 2J02). The domain V region of 23S rRNA has been shown in grey and the 3′-CCA end of P-site tRNA is represented in red. The nucleotides which have been mutated in this study have also been shown in sticks and are represented in the same colour as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101293#pone-0101293-g004" target="_blank">Fig. 4A</a>. (<b>B</b>) The 23S rRNA of <i>E. coli</i> large ribosomal subunit (PDB: 2I2V) has also been displayed (light orange) here with P-site tRNA (PDB: 2J02) (red colour surface representation). This view is in the same orientation in which Fig. 5A has been shown.</p

Effects of mutation on bDV RNA assisted BCAII reactivation.

<p>(<b>A</b>) The secondary structure of the bDV RNA has been shown here. The mutated nucleotides are also shown here using colour code. Nucleotides that are the binding sites of antibiotics blasticidin (b), puromycin (p), josamycin (j) and erythromycin (e) are also shown in the structure. The five specific nucleotides of bDV RNA which interacts with unfolded protein are also indicated by arrows. (<b>B</b>) Reactivation of BCAII in the presence of wild type (wt) and mutants <i>E. coli</i> bDV RNA. The reactivation of BCAII with mutated and wt bDV RNA was represented as bar diagrams. Mutated bDV RNAs are marked as M1 to M9 (M1- A2602C, M2- delA2602, M3- U2585C, M4- delU2585, M5- G2252C, M6- delG2252, M7- A2451G, M8- delA2451, M9- G2553C). BCAII reactivation in absence of bDV RNA is marked as ‘Self’ and in presence of wt bDV RNA is marked as ‘bDV RNA’. (<b>C</b>) Time course of binding of [α- ³²P] labelled RNA1 to refolding BCAII. Unfolded BCAII was refolded in presence of [α- ³²P] labelled wt RNA1 or mutant RNA1. Aliquots of samples were withdrawn at different time points from the RNA1-refolding protein mix and filtered through nitrocellulose. Percent (%) radioactivity retained (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101293#s2" target="_blank">Materials and Methods</a>) on the filter for wtRNA1 (Δ) and its mutants G2553C (□), A2602C (◊) and U2585C (○) are plotted against time. Time course of RNA2 mediated release of [α- ³²P] labelled RNA1 from RNA1-BCAII complex. The [α- ³²P] labelled RNA1 (wild type or mutated) was incubated with refolding BCAII for 5 min, to which RNA2 was added. Equal volume of samples were withdrawn at different time points and filtered through nitrocellulose. Percent (%) radioactive (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101293#s2" target="_blank">materials and methods</a>) retained on the filter for wtRNA1 + wtRNA2 (▪), wtRNA1 + G2252CRNA2 (▾), wtRNA1 + delG2252RNA2 (♦), U2585CRNA1 + wtRNA2 (•), G2553CRNA1 + wtRNA2 (▴) are plotted against time.</p

Effects of antibiotics on ribosome and domain V RNA assisted BCAII reactivation.

<p>(<b>A</b>) Dose dependent inhibition of ribosome's chaperoning ability due to binding antibiotics blasticidin and puromycin. <i>E. coli</i> ribosome was allowed to bind to increasing concentrations of the antibiotics in their respective buffers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101293#pone.0101293.s003" target="_blank">Table S1</a>). Reactivation of BCAII by ribosome-antibiotic complexes with antibiotics blasticidin (▪) and puromycin (•) are plotted against the log [antibiotics] (as indicated in x-axis of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101293#pone-0101293-g003" target="_blank">Fig. 3A</a>). The refolding experiments were repeated at least 3 times and the average values were plotted. The graph was fitted by Boltzmann fit. (<b>B</b>) Dose dependent inhibition of ribosome's chaperoning ability due to binding of macrolide antibiotics. The ribosome was allowed to bind to erythromycin or josamycin in their respective buffers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101293#pone.0101293.s003" target="_blank">Table S1</a>). Reactivation of BCAII by ribosome-antibiotic complexes with antibiotics erythromycin (▪) and josamycin (•) bound ribosome were plotted against the log [antibiotics] (as indicated in x-axis of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101293#pone-0101293-g003" target="_blank">Fig. 3B</a>).The refolding experiments were repeated at least 3 times and the average values were plotted. The graph was fitted by Boltzmann distribution fit. (<b>C</b>) A comparison of BCAII reactivation in presence of bDV RNA and bDV RNA complexed with total <i>E. coli</i> tRNA (15 µM), blasticidin (BLS; 3 mM), puromycin (PURO; 6 mM), erythromycin (ERY; 1.5 mM) and josamycin (JOSA; 2 mM) are shown in the bar diagram.</p

Effects of tRNA on programmed ribosome's chaperoning ability.

<p>(<b>A</b>) Schematic design of template for in vitro transcription of mRNA. The double stranded DNA was designed such that the ribosome binding site would position the methionine (AUG) and glutamic acid (GAA) codon at P-site and A-site respectively. These sequences are downstream of a T7 promoter sequence that was used for transcription of mRNA by T7 RNA polymerase (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101293#s2" target="_blank">Materials and Methods</a>). (<b>B</b>) Dose dependent inhibition of ribosome's chaperoning ability due to binding of tRNA. The <i>E. coli</i> ribosome programmed with mRNA was allowed to bind to increasing concentrations of Met- tRNA (▪) and total <i>E. coli</i> tRNA (•) in Buffer-P (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101293#pone.0101293.s003" target="_blank">Table S1</a>). Inhibition of chaperoning activity of nonprogrammed ribosome (▴) in presence of increasing concentration of Met-tRNA is also plotted here. Reactivation (%) of BCAII by ribosome-tRNA complex was plotted against its increasing tRNA concentrations. The refolding experiments were repeated at least 3 times and the average values were plotted. The graph was fitted by Boltzmann fit. (<b>C</b>) Effect of presence of both deacylated Met-tRNA and Glu-tRNA on chaperoning ability of programmed ribosome. Bar diagram shows BCAII reactivation by the complexes 2 to 6; programmed ribosome (70S ribosome + mRNA) (2), Glu-tRNA bound programmed ribosome (70S ribosome + mRNA +1.5 µM Glu-tRNA) (3) and Met-tRNA bound programmed ribosome (70S ribosome + mRNA +0.75 µM Met-tRNA) (4). BCAII reactivation by complex 4 upon further addition of 0.75 µM of Glu-tRNA (5) or 70S ribosome + mRNA +1.5 µM Met-tRNA (6) is also shown. (<b>D</b>) Bar diagram represents a comparison of the BCAII reactivation (%) of; Self (1), 70S ribosome + mRNA (2), 70S ribosome + mRNA+1.5 µM Met-tRNA (3), 70S ribosome + mRNA + tetracyclin (4), 70S ribosome + mRNA+ tetracyclin +1.5 µM Met-tRNA (5).</p

The Ribosome Can Prevent Aggregation of Partially Folded Protein Intermediates: Studies Using the <i>Escherichia coli</i> Ribosome

<div><p>Background</p><p>Molecular chaperones that support de novo folding of proteins under non stress condition are classified as chaperone ‘foldases’ that are distinct from chaperone’ holdases’ that provide high affinity binding platform for unfolded proteins and prevent their aggregation specifically under stress conditions. Ribosome, the cellular protein synthesis machine can act as a foldase chaperone that can bind unfolded proteins and release them in folding competent state. The peptidyl transferase center (PTC) located in the domain V of the 23S rRNA of <i>Escherichia coli</i> ribosome (bDV RNA) is the chaperoning center of the ribosome. It has been proposed that via specific interactions between the RNA and refolding proteins, the chaperone provides information for the correct folding of unfolded polypeptide chains.</p><p>Results</p><p>We demonstrate using <i>Escherichia coli</i> ribosome and variants of its domain V RNA that the ribosome can bind to partially folded intermediates of bovine carbonic anhydrase II (BCAII) and lysozyme and suppress aggregation during their refolding. Using mutants of domain V RNA we demonstrate that the time for which the chaperone retains the bound protein is an important factor in determining its ability to suppress aggregation and/or support reactivation of protein.</p><p>Conclusion</p><p>The ribosome can behave like a ‘holdase’ chaperone and has the ability to bind and hold back partially folded intermediate states of proteins from participating in the aggregation process. Since the ribosome is an essential organelle that is present in large numbers in all living cells, this ability of the ribosome provides an energetically inexpensive way to suppress cellular aggregation. Further, this ability of the ribosome might also be crucial in the context that the ribosome is one of the first chaperones to be encountered by a large nascent polypeptide chains that have a tendency to form partially folded intermediates immediately following their synthesis.</p></div

Effects of bDV RNA mutants on refolding of BCAII.

<p>A) Bar diagram shows percent aggregation reduction during BCA II-m refolding by bDV RNA, mDV RNA and bDV RNA mutants U2585C (UC), delG2252 (delG). The turbidities in each case were measured at 320 nm, 12 minutes after return to refolding conditions and turbidity in absence of chaperone was assumed as 100%. B) Comparison of reactivation yields of BCAII-m (0.3 µM) after 30 min of refolding in absence of the chaperone (Self) and in presence of bDV RNA1(R1), bDV RNA1+RNA2 (R1+R2), RNA1+3% Ethanol (R1+EtOH), del G2252 bDV RNA (delG), del G2252+ RNA2 (delG+R2), U2585C (UC), U2585C bDV RNA+RNA2 (UC+R2) and U2585C bDV RNA+3% Ethanol (UC+EtOH). C) Comparison of the reactivation yields of BCAII-m (0.9 µM) after 30 min of refolding in absence of the chaperone (Self), in presence of del G2252 bDV RNA and mDV RNA. BCAII-m reactivation upon addition of RNA 2 portion of bDV at zero minute (mDV+R2 0′and delG+R2 0′) and after ten minutes of initiation of refolding (mDV+R2 10′ and delG+R2 10′) are also shown.</p

Interaction of BCAII and lysozyme with ribosome and its domain V RNA.

<p><i>Filter binding studies.</i> Refolding of BCAII-m or reduced-denatured lysozyme was initiated in presence of radiolabeled various domain V RNA, was UV- crosslinked and filtered through nitrocellulose membrane (material and method). A) Time course of interactions of BCAII-m with radiolabeled bDV RNA (-○-), mDV RNA (-▴-), bDV RNA mutants U2585C (-▾-) and delG2252 (-▪-) are shown here. Experiments were repeated thrice and their average values were taken for final data plotting. B) Time course of interactions of reduced-denatured lysozyme and radiolabeled bDV RNA (-•-) and mDV RNA (-▴-) are shown. <i>Size exclusion chromatography.</i> Refolding of FITC labeled BCAII-m or reduced-denatured lysozyme was initiated in presence of 70S ribosome, was UV-crosslinked at 30 second of refolding, and the mix was loaded on Sephacryl S-300 column. The elution of the protein and the ribosome was monitored by fluorescence at 518 nm and absorbance at 260 nm. C) Detection of 70S- BCAII complex. The elution profiles of BCAII-m in presence of ribosome at 30 seconds of refolding (3), reduced denatured lysozyme in presence of ribosome at 30 seconds of refolding (5) are shown. The elution profiles of ribosome (1), native BCAII (2) and native lysozyme (4) are also shown for comparison.</p