Journal: PLoS Pathogens
Article Title: Biochemical and Structural Insights into the Mechanisms of SARS Coronavirus RNA Ribose 2?-O-Methylation by nsp16/nsp10 Protein Complex
Figure Lengend Snippet: Biochemical analyses of the MTase activities of SARS-CoV nsp14, nsp16 and nsp10. (A) TLC analysis of nuclease P1-resistant cap structures released from 32 P-labeled G*pppA-RNA methylated by nsp10, nsp14, nsp16, nsp14/nsp10, nsp16/nsp10, nsp14/nsp16, and nsp10/nsp14/nsp16, respectively (lanes 1–7), and m7G*pppA-RNA methylated by nsp16/nsp10 (at pH 7.5), nsp10, nsp14, nsp16, nsp14/nsp10, nsp10/nsp14/nsp16, nsp16/nsp10, and nsp14/nsp16, respectively (at pH 8.0) (lanes 10–17). The markers G*pppA (lane 8), m7G*pppA (lane 9), and m7G*pppAm (lane 18) were prepared with commercial vaccinia virus capping enzymes. The positions of origin and migration of G*pppA, m7G*pppA, and m7G*pppAm (lanes 8, 9, and 18) are indicated on the left. The bands located between origin and G*pppA are free α- 32 P-GTP, which may be left over after one-step purification of labeled RNA. (B) Different RNA substrates were used to test the methylation activities of nsp16/nsp10 complex of SARS-CoV ( n = 3, mean values ± SD). (C) 32 P-labeled G*pppG-RNA was used as substrate to test the methylation activities of nsp16/nsp10, nsp16, and nsp10 respectively. Vaccinia VP39 2′-O-MTase was used as a positive control ( n = 3, mean values ± SD).
Article Snippet: Biochemical assays for MTase activity Purified recombinant or mutant proteins (0.5 µg) and 2×103 cpm of 32 P-labeled m7G*pppA-RNA or G*pppA-RNA substrates were added to 8.5 µL reaction mixture [40 mM Tris-HCl (pH 7.5 or 8.0), 2 mM MgCl2 , 2 mM DTT, 10 units RNase inhibitor, 0.2 mM SAM] and incubated at 37°C for 1.5 h. RNA cap structures were liberated with 5 µg of nuclease P1 (Sigma), then spotted onto polyethyleneimine cellulose-F plates (Merck) for TLC, and developed in 0.4 M ammonium sulfate.
Techniques: Thin Layer Chromatography, Labeling, Methylation, Migration, Purification, Positive Control