Complementary Role of Two Fragments of Domain V of 23 S Ribosomal RNA in Protein Folding

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.

Section: Domain V Of the 23s/25s/28s Rrna Of The Large Ribosomal Subumentioning

confidence: 99%

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

Pang

Kurella

Voisset

et al. 2013

“…4, compare A with B and C). We have shown earlier that the bacterial PT loop is responsible for folding the protein to a competent state, and the RNA2 region is required to release it (21). Taking the cue from these, we added bacterial RNA2 in the refolding reaction to see whether it could supplement in the folding of the denatured protein by mitochondrial PTC (21).…”

Section: Refolding Of Denatured Bca By Bovine Mitochondrial Ptc Rna Cmentioning

confidence: 99%

“…BCAII, lactate dehydrogenase (LDH), porcine heart mitochondrial malate dehydrogenase (MDH), and human carbonic anhydrase I (HCAI) were denatured with guanidine hydrochloride (25)(26)(27), and the loss of their secondary structures was verified by CD spectra (19,21). For all the enzymes 6 M guanidine hydrochloride was used for denaturation.…”

Section: Denaturation and Refolding Of Proteinsmentioning

confidence: 99%

“…5 The structure-function relationship of this RNA segment became easier to study when we could split bacterial domain V into the more conserved central loop region (RNA1, the PTC) and the highly variable stem-loop region (RNA2) outside the central loop. The RNA1 transforms an unfolded protein to a FCS, and the RNA2 part is required to dissociate the folding-competent state (FCS) from RNA1 (21).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Involvement of Mitochondrial Ribosomal Proteins in Ribosomal RNA-mediated Protein Folding

Das

Ghosh

Bhattacharya

et al. 2011

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.Discovery of the existence of relics of the RNA world such as "ribozymes," where the RNA component comprised the active principle and could provide catalytic function by itself in vitro, strengthened a RNA-world hypothesis. A corollary of the RNA world model is that with time RNAs would have to be gradually replaced by proteins as catalysts due to their greater flexibility in conformations that gives them the versatility needed for varied catalytic properties (1-6). Protein catalysts generally have much higher turnover numbers (shorter reaction times) but not necessarily lower K m values than their RNA counterparts (5, 7). So it may be interesting to find relics of such isofunctional activities in biology.Ribosomes have been identified as a general protein folding modulator on the basis of their ability to successfully fold all the denatured and newly synthesized proteins studied so far both in vitro and in vivo (8 -20). For each unfolded protein, the level of recovery in activity with ribosome is around 80 -100%. The protein folding activity has been found to reside in the domain V of the 23 S rRNA in 50 S subunit of the bacterial ribosome and its homolog elsewhere, as in those from plant and animal cytosols. This domain has long been known to harbor the peptidyl transferase center (PTC).5 The structure-function relationship of this RNA segment became easier to study when we could split bacterial domain V into the more conserved central loop region (RNA1, the PTC) and the highly variable stem-loop region (RNA2) outside the central loop. The RNA1 transforms an unfolded protein to a FCS, and the RNA2 part is required to dissociate the folding-competent state (FCS) from RNA1 (21).In mitochondrial ribosome (mitoribosome) in a number of organisms, the conserved central loop of the peptidyl transferase center (corresponding to bacterial RNA1 part) is present, but the variable stem loop region (corresponding to the bacterial RNA2 region) is almost completely deleted. Here we report that the mitoribosomal PTC is an efficient protein folding modulator like the prokaryotic and eukaryotic cytosolic ribosomes, and mitoribosomal proteins compensate fo...

“…These results suggest that additional cellular components may be required for the successful folding of these polypeptide chains in vivo. The recent discovery that the ribosome-associated trigger factor may cooperate with the molecular chaperone DnaK to assist in the folding of some polypeptide chains (16,17) suggests that additional parts of the cellular folding machinery may lie very close to the ribosome (18) and may involve the ribosome itself (19,20).…”

mentioning

confidence: 99%

A Newly Synthesized, Ribosome-bound Polypeptide Chain Adopts Conformations Dissimilar from Early in VitroRefolding Intermediates

Clark

King

2001

Little is known about the conformations of newly synthesized polypeptide chains as they emerge from the large ribosomal subunit, or how these conformations compare with those populated immediately after dilution of polypeptide chains out of denaturant in vitro. Both in vivo and in vitro, partially folded intermediates of the tailspike protein from Salmonella typhimurium phage P22 can be trapped in the cold. A subset of monoclonal antibodies raised against tailspike recognize partially folded intermediates, whereas other antibodies recognize only later intermediates and/or the native state. We have used a pair of monoclonal antibodies to probe the conformational features of full-length, newly synthesized tailspike chains recovered on ribosomes from phage-infected cells. The antibody that recognizes early intermediates in vitro also recognizes the ribosome-bound intermediates. Surprisingly, the antibody that did not recognize early in vitro intermediates did recognize ribosome-bound tailspike chains translated in vivo. Thus, the newly synthesized, ribosome-bound tailspike chains display structured epitopes not detected upon dilution of tailspike chains from denaturant. As opposed to the random ensemble first populated when polypeptide chains are diluted out of denaturant, folding in vivo from the ribosome may begin with polypeptide conformations already directed toward the productive folding and assembly pathway.