Department of Cell and Molecular Biology, Uppsala University, 75124 Uppsala, Sweden.
J Biol Chem. 2013 Jun 28;288(26):19081-9. doi: 10.1074/jbc.M113.466748. Epub 2013 May 14.
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.
核糖体大亚基的 23S/25S/28S rRNA 结构域 V 构成了核糖体蛋白折叠活性（PFAR）的活性中心。本研究使用来自大肠杆菌和酿酒酵母的体外转录的结构域 V rRNA 作为折叠调节剂，以人碳酸酐酶作为模型蛋白，证明了从原核生物到真核生物 PFAR 都是保守的。先前的研究表明，抗朊病毒化合物 6-氨基菲啶（6AP）抑制 PFAR。本研究采用 UV 交联和引物延伸实验，证明了蛋白质底物和 6AP 与 23S rRNA 结构域 V 上的一组共同核苷酸相互作用。在相互作用位点发生突变会降低 PFAR，并导致蛋白质底物和 6AP 的结合模式发生改变或丧失。此外，人碳酸酐酶重折叠的动力学分析表明，6AP 降低了重折叠蛋白的产量，但不影响重折叠的速率。因此，我们得出结论，6AP 竞争性地阻止蛋白质底物与 rRNA 结合，从而抑制 PFAR。最后，我们提出了一个阐明 6AP 抑制 PFAR 的机制的方案。
