Abstract
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.
Highlights
It has been shown over the past 2 decades that the ribosome is able to refold ϳ20 different proteins in vitro [1,2,3,4,5]
Refolding of HCA with Domain V rRNAs—HCA denatured in 6 M guanidine hydrochloride was subjected to refolding in the presence of in vitro transcribed domain V of 23S rRNA from E. coli and 25S rRNA from S. cerevisiae
6AP-mediated Inhibition of the Protein Folding Activity of Domain V of 23S rRNA—Because domain V of rRNA constitutes the active center for protein folding activity of the ribosome (PFAR) [7], we studied the mode of action of 6AP as an inhibitor using domain V of 23S rRNA as a folding modulator
Summary
Proteins—Human carbonic anhydrase (HCA) was expressed in E. coli and purified by column chromatography as described [20]. UV Cross-linking— 6 M guanidine hydrochloride-denatured proteins (HCA, bovine carbonic anhydrase, and dihydrofolate reductase, all at 30 M) were diluted 100 times in refolding buffer containing domain V of 23S rRNA from E. coli (300 nM), and UV cross-linking was performed immediately in a Bio-Rad GS Gene LinkerTM instrument, with 254 nm UV irradiation (600 mJ) [21]. For cross-linking with 6AP, 300 nM domain V of 23S rRNA and 0.5 mM 6AP (or 6APi) were mixed and subjected to the same procedure as described above In both cases, the samples were kept on ice during irradiation to prevent heat damage of the RNA. The products were precipitated, washed with 70% ethanol, and run on a 6.5% polyacrylamide gel with 8 M urea next to a sequencing ladder of domain V rDNA obtained using the same primer by Thermo Sequenase DNA polymerase (Thermo SequenaseTM sequencing kit, USB Corp.)
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