Identical RNA-Protein Interactions in Vivo and in Vitro and a Scheme of Folding the Newly Synthesized Proteins by Ribosomes

Das, Debasis; Samanta, Dibyendu; Hasan, Syed; Das, Anindita; Bhattacharya, Arpita; Dasgupta, Santanu; Chakrabarti, Abhijit; Ghorai, Pradip Kr.; Gupta, Chanchal Das

doi:10.1074/jbc.m112.396127

Cited by 12 publications

(23 citation statements)

References 49 publications

(31 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Binding Sites of Three Protein Substrates on Domain V of 23S rRNA-The protein-binding sites on the central loop and lower part of domain V of 23S rRNA have been reported previously for bovine carbonic anhydrase, lysozyme, malate dehydrogenase, and lactate dehydrogenase protein substrates using UV cross-linking followed by primer extension (21,23). We extended these observations using HCA and dihydrofolate reductase as protein substrates.…”

Section: Resultssupporting

confidence: 56%

The Antiprion Compound 6-Aminophenanthridine Inhibits the Protein Folding Activity of the Ribosome by Direct Competition

Pang

Kurella

Voisset

et al. 2013

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Background: 6-Aminophenanthridine (6AP) is an inhibitor of the protein folding activity of the ribosome (PFAR). Results: The protein substrates and 6AP bind at common sites on rRNA; mutations at those sites abolish binding and inhibit PFAR. Conclusion: 6AP competitively obstructs the protein-binding sites and thereby inhibits PFAR. Significance: We have clarified the mechanism by which 6AP inhibits PFAR.

show abstract

Section: Resultssupporting

confidence: 56%

The Antiprion Compound 6-Aminophenanthridine Inhibits the Protein Folding Activity of the Ribosome by Direct Competition

Pang

Kurella

Voisset

et al. 2013

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Toe-printing and MALDI-ToF MS/MS studies revealed that the 23SrRNA interacts with a nascent protein through the same nucleotides as it does in vitro (in presence of Mg 2+ ) with the chemically unfolded form of the same protein when added “in trans” to 70S [12, 19]. A number of unfolded proteins, e.g., bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., have been shown to interact with the same nucleotides as well.…”

Section: Resultsmentioning

confidence: 99%

“…Albeit a number of studies [9, 10] showed interaction of nascent protein with the wall of the peptide exit tunnel, leaving the possibility of slow release of nascent polypeptide from the ribosome. A full length nascent protein, tagged by C-terminal His, isolated from growing bacterial cell as well as from in vitro translation reaction was found associated predominantly with the 50S subunits, and a little with the 70S [11, 12]. Nucleotides of 23SrRNA interacting with that nascent protein are present in the close proximity of conserved inter-subunit bridge B2a/B2b joining the 50S and 30S [12].…”

Section: Introductionmentioning

confidence: 99%

“…A full length nascent protein, tagged by C-terminal His, isolated from growing bacterial cell as well as from in vitro translation reaction was found associated predominantly with the 50S subunits, and a little with the 70S [11, 12]. Nucleotides of 23SrRNA interacting with that nascent protein are present in the close proximity of conserved inter-subunit bridge B2a/B2b joining the 50S and 30S [12]. The fact that RRF also interact at the same site [13, 14]may indicate that access of those nucleotides for RRF is only possible once nascent protein leaves the 70S; however isolation of predominant 50S population bound to full length nascent protein in fact lead us to question its role together with that of RRF in dissociating the 70S ribosome.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2017

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…Hence, most of the analysis of the mechanism of ribosome's chaperoning function that has been reported in literature has been performed on the in vitro transcribed bDV RNA [12], [13], [28]. The effects of tRNA and antibiotics on the chaperoning activity of this RNA domain were therefore studied using denatured BCAII as the substrate protein.…”

Section: Methodsmentioning

confidence: 99%

Impact of P-Site tRNA and Antibiotics on Ribosome Mediated Protein Folding: Studies Using the Escherichia coli Ribosome

2014

View full text Add to dashboard Cite

BackgroundThe 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.ResultsThe 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.ConclusionBoth 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.

show abstract

Identical RNA-Protein Interactions in Vivo and in Vitro and a Scheme of Folding the Newly Synthesized Proteins by Ribosomes

Cited by 12 publications

References 49 publications

The Antiprion Compound 6-Aminophenanthridine Inhibits the Protein Folding Activity of the Ribosome by Direct Competition

The Antiprion Compound 6-Aminophenanthridine Inhibits the Protein Folding Activity of the Ribosome by Direct Competition

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

Impact of P-Site tRNA and Antibiotics on Ribosome Mediated Protein Folding: Studies Using the Escherichia coli Ribosome

Contact Info

Product

Resources

About