We have shown that the domain V of bacterial 23 S rRNA could fold denatured proteins to their active state. This segment of 23 S rRNA could further be split into two parts. One part containing mainly the central loop of domain V could bind denatured human carbonic anhydrase I stably. This association could be reversed by adding the other part of domain V. The released enzyme was directed in such a way by the central loop of domain V that it could now fold by itself to active form. This agrees with our earlier observation that proteins fold within the cell posttranslationally, a process that is completed after release of the newly synthesized polypeptide from the ribosome (Chattopadhyay, S., Pal, S., Chandra, S., Sarkar, D., and DasGupta, C. (1999) Biochim. Biophys. Acta 1429, 293-298).

S PAL

S CHANDRA

S CHOWDHURY

D SARKAR

A N GHOSH

GUPTA C DAS

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University of Calcutta (informally known as Calcutta University or CU) is a collegiate public state university located in Kolkata, West Bengal, India.&nbsp;Within India it is recognized as a &quot;Five-Star University&quot; and accredited &quot;A&quot; Grade by National Assessment and Accreditation Council.

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The University of Calcutta has secured the Fifth position among the Universities of India in the prestigious &quot;Indian University Ranking 2019&quot; list, released by the NIRF of the Ministry of Human Resource Development, Government of India.

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It was established on 24 January 1857, and was one of the first institutions in Asia to be established as a multidisciplinary and Western-style university. Its alumni and faculty include several heads of state, heads of government, social reformers, prominent artists, only academy award winner and Dirac medal winner in India, many Fellows of the Royal Society and five Nobel laureates as of 2019 which is highest in South Asia.

University Of Calcutta

Journal of Biological Chemistry

Background: The ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is also capable of assisting in folding of polypeptide chains. The peptidyl transferase center (PTC) that catalyzes peptide bond formation resides in the domain V of the 23S rRNA of the bacterial ribosome. Proper positioning of the 3′ -CCA ends of the A- and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is also the center for ribosomal chaperoning activity. The unfolded polypeptide chains interact with the specific nucleotides of the PTC and are released in a folding competent form. In vitro transcribed RNA corresponding to this domain (bDV RNA) also displays chaperoning activity. Results: The present study explores the effects of tRNAs, antibiotics that are A- and P-site PTC substrate analogs (puromycin and blasticidin) and macrolide antibiotics (erythromycin and josamycin) on the chaperoning ability of the E. coli ribosome and bDV RNA. Our studies using mRNA programmed ribosomes show that a tRNA positioned at the P-site effectively inhibits the ribosome's chaperoning function. We also show that the antibiotic blasticidin (that mimics the interaction between 3′-CCA end of P/P-site tRNA with the PTC) is more effective in inhibiting ribosome and bDV RNA chaperoning ability than either puromycin or the macrolide antibiotics. Mutational studies of the bDV RNA could identify the nucleotides U2585 and G2252 (both of which interact with P-site tRNA) to be important for its chaperoning ability. Conclusion: Both protein synthesis and their proper folding are crucial for maintenance of a functional cellular proteome. The PTC of the ribosome is attributed with both these abilities. The silencing of the chaperoning ability of the ribosome in the presence of P-site bound tRNA might be a way to segregate these two important functions. © 2014 Mondal et al.

PLoS ONE

Impact of P-Site tRNA and antibiotics on ribosome mediated protein folding: Studies using the Escherichia coli ribosome

Domain V of the 23S/25S/28S rRNA of the large ribosomal subunit constitutes the active center for the protein folding activity of the ribosome (PFAR). Using in vitro transcribed domain V rRNAs from Escherichia coli and Saccharomyces cerevisiae as the folding modulators and human carbonic anhydrase as a model protein, we demonstrate that PFAR is conserved from prokaryotes to eukaryotes. It was shown previously that 6-aminophenanthridine (6AP), an antiprion compound, inhibits PFAR. Here, using UV cross-linking followed by primer extension, we show that the protein substrates and 6AP interact with a common set of nucleotides on domain V of 23S rRNA. Mutations at the interaction sites decreased PFAR and resulted in loss or change of the binding pattern for both the protein substrates and 6AP. Moreover, kinetic analysis of human carbonic anhydrase refolding showed that 6AP decreased the yield of the refolded protein but did not affect the rate of refolding. Thus, we conclude that 6AP competitively occludes the protein substrates from binding to rRNA and thereby inhibits PFAR. Finally, we propose a scheme clarifying the mechanism by which 6AP inhibits PFAR. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

The antiprion compound 6-aminophenanthridine inhibits the protein folding activity of the ribosome by direct competition

Background: Ribosomal PTC acts as a protein folding modulator in vivo and in vitro. Results: A fixed set of nucleotides in the PTC interacts to fold polypeptides in vivo and in vitro. Conclusion: Folding all proteins through interaction with the same set of nucleotides in PTC implies they have intrinsic homology. Significance: Hundreds of proteins showed an identical cumulative hydrophobicity plot for amino acids. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Identical RNA-protein interactions in vivo and in vitro and a scheme of folding the newly synthesized proteins by ribosomes

The peptidyl transferase center of the domain V of large ribosomal RNA in the prokaryotic and eukaryotic cytosolic ribosomes acts as general protein folding modulator. We showed earlier that one part of the domain V (RNA1 containing the peptidyl transferase loop) binds unfolded protein and directs it to a folding competent state (FCS) that is released by the other part (RNA2) to attain the folded native state by itself. Here we show that the peptidyl transferase loop of the mitochondrial ribosome releases unfolded proteins in FCS extremely slowly despite its lack of the rRNA segment analogous to RNA2. The release of FCS can be hastened by the equivalent activity of RNA2 or the large subunit proteins of the mitochondrial ribosome. The RNA2 or large subunit proteins probably introduce some allosteric change in the peptidyl transferase loop to enable it to release proteins in FCS. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Involvement of mitochondrial ribosomal proteins in ribosomal RNA-mediated protein folding

The rate of protein synthesis is about seven and fifteen amino acids per second, in the eukaryotic and the bacterial ribosome, respectively. Hence, a few minutes is required to synthesize a polypeptide of an average length. This is much longer than the time needed for the hydrophobic collapse (folding) to take place. So a polypeptide gets enough time to form its local secondary to tertiary structures cotranslationally and put such segments in proper order while in association with the ribosome, unless something prevents its entire length from folding. As reported earlier, ribosomes from prokaryotes, eukaryotes, and mitochondria act as molds for protein folding, and each mold has a set of recognition sites for all proteins. More specifically, the mold is the peptidyl transferase center (PTC), a part of the large RNA of the large ribosomal subunit. Specific amino acids from different random coil regions in a protein interact with specific nucleotides in the PTC, which brings the entire length of the protein into the small space of the PTC mold. The mold thus helps to stabilize the entropy-driven collapsed state of the polypeptide. The process also divides the protein into small segments; each segment is connected at two ends with two nucleotides and can fold in the ribosomal environment. The segments dissociate in such a sequence that the organization proceeds hierarchically from the core of the globular protein radially towards the outer surface. Then the protein dissociates from the ribosome in a "folding competent state" which does the final fine tuning in folding outside the ribosome. While the ribosomal contact and release are over in 1-2 minutes in vitro, the fine tuning takes about 5-10 minutes. Release from the ribosome needs no added energy factor from outside, like ATP. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Israel Journal of Chemistry

Ribosome: The structure-function relation and a new paradigm to the protein folding problem

In vitro transcripts containing domain V of the 23S rRNA of Escherichia coli and Bacillus subtilis can reactivate denatured proteins almost as efficiently as the total 23S rRNA. Here we show that almost the full length of domain V is required for reactivation of denatured pig muscle lactate dehydrogenase and pig heart cytoplasmic malate dehydrogenase: the central loop of this domain alone is not enough for this purpose. The antibiotic chloramphenicol, which binds to domain V of 23S rRNA, can inhibit reactivation of these proteins completely. Activity is eliminated by EDTA at a concentration of &lt; 1 mM, even in the presence of 4 mM MgCl2, suggesting that the three-dimensional conformation of the RNA should be maintained for this activity.

Nucleic Acids Research

Reactivation of denatured proteins by domain V of bacterial 23S rRNA

Escherichia coli ribosome, its 50S subunit, or simply the 23S rRNA can reactivate denatured proteins in vitro. Here we show that protein synthesis inhibitors chloramphenicol and erythromycin, which bind to domain V of 23S rRNA of E. coli, can inhibit reactivation of denatured pig muscle lactate dehydrogenase and fungal glucose-6-phosphate dehydrogenase by 23S rRNA completely. Oligodeoxynucleotides complementary to two regions within domain V (which cover sites of chloramphenicol resistant mutations and the putative A site of the incoming aminoacyl tRNA), but not to a region outside of domain V, also can inhibit the activity. Domain V of 23S rRNA, therefore, appears to play a crucial role in reactivation of denatured proteins.

Proceedings of the National Academy of Sciences of the United States of America

Reactivation of denatured proteins by 23S ribosomal RNA: Role of domain V

Ribosomes from a number of prokaryotic and eukaryotic sources (e.g., Escherichia coli, wheat germ and rat liver) can refold a number of enzymes which are denatured with guanidine/HCl prior to incubation with ribosomes. In this report, we present our observations on the refolding of denatured lactate dehydrogenase from rabbit muscle and glucose-6-phosphate dehydrogenase from baker's yeast by ribosomes from E. coli, wheat germ and rat liver. The protein-folding acivity of E. coli ribosomes was found to be present in 50S particles and in 23S rRNA. The 30S particle or 16S rRNA did not show any protein-folding activity. The protein-folding activity of 23S rRNA may depend on its tertiary conformation. Loss of tertiary structure, by incubation with low concentrations of EDTA, inhibited the protein-folding activity of 23S rRNA. This low concentration of EDTA had no effect on folding of the denatured enzymes by themselves.

European Journal of Biochemistry

In vitro protein folding by ribosomes from Escherichia coli, wheat germ and rat liver: The role of the 50S particle and its 23S rRNA

Escherichia coli ribosomes were used to refold denatured lactate dehydrogenase from porcine muscle. This activity of ribosomes, unlike most of the chaperons, did not require the presence of ATP. The molar concentration of ribosomes required for this refolding was comparable with that of the enzyme. Restoration of the enzyme activity was demonstrated using assays for both the forward and backward reactions. Binding of the denatured enzyme to ribosomes and its refolding were fairly rapid processes as revealed by the time course of the reaction and inhibition of folding when the denatured enzyme was allowed to refold spontaneously for short times before the addition of ribosomes. This protein-folding activity was detected in 70 S ribosomes as well as its RNA, in 50 S particles and in 23 S rRNA. However, 30 S particles failed to refold the enzyme.

Journal	Journal of Biological Chemistry
Publisher	AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
ISSN	0021-9258
Open Access	Yes