• BMB Reports
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• v.40 no.5
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• pp.649-655
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• 2007

The nsp14 protein is an exoribonuclease that is encoded by severe acute respiratory syndrome coronavirus (SARS-CoV). We have cloned and expressed the nsp14 protein in Escherichia coli, and characterized the nature and the role(s) of the metal ions in the reaction chemistry. The purified recombinant nsp14 protein digested a 5'-labeled RNA molecule, but failed to digest the RNA substrate that is modified with fluorescein group at the 3'-hydroxyl group, suggesting a 3'-to-5' exoribonuclease activity. The exoribonuclease activity requires $Mg^{2+}$ as a cofactor. Isothermal titration calorimetry (ITC) analysis indicated a two-metal binding mode for divalent cations by nsp14. Endogenous tryptophan fluorescence and circular dichroism (CD) spectra measurements showed that there was a structural change of nsp14 when binding with metal ions. We propose that the conformational change induced by metal ions may be a prerequisite for catalytic activity by correctly positioning the side chains of the residues located in the active site of the enzyme.

• Korean Journal of Microbiology
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• v.43 no.1
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• pp.72-75
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• 2007

Using co-immunoprecipitation, we identified proteins interacting with Streptomyces coelicolor RNase ES, an ortholog of Escherichia coli RNase E that plays a major role in RNA decay and processing. Polyphosphate kinase and a homolog of exoribonuclease polynucleotide phosphorylase, guanosine pentaphosphate synthetase I that use inorganic phophate were co-precipitated with RNase E, indicating a possibility of S. coelicolor RNase ES to form a multiprotein complex called degradosome, which has been shown to be formed by RNase E in E. coli. Polynucleotide phophorylase proteins from these two phylogenetically distantly related bacteria species showed similar RNA cleavage action in vitro. These results imply the ability of RNase ES to form a multiprotein complex that has structurally and functionally similar to that of E. coli degradosome.