Journal: Journal of Virology
Article Title: Nuclear Magnetic Resonance Structure of the N-Terminal Domain of Nonstructural Protein 3 from the Severe Acute Respiratory Syndrome Coronavirus ▿
Figure Lengend Snippet: Association of nsp3a(1-183) and nsp3a(1-112) purified from E. coli with nucleic acids. (a) Nucleic acid was visualized with SYBR-gold staining before or after digestion with nucleases specific to DNA (DNase I or T7 endonuclease) or RNA (RNase I, RNase A, or RNase T 1 ). Cleavage assays were performed at 37°C for 1 h, and digested samples were analyzed by native electrophoresis on precast 6% polyacrylamide gels. Open arrowheads denote copurified nucleic acid species associated with nsp3a(1-112) or nsp3a(1-183), respectively. (b) EMSAs were performed to estimate the RNA binding affinity of nsp3a(1-112). Samples containing ssRNA1 or ssRNA2 were incubated at 37°C for 1 h with variable concentrations of protein and analyzed by native electrophoresis on precast 6% polyacrylamide gels. RNA was detected by SYPRO-gold poststain, and the fraction of bound RNA was calculated relative to the maximum binding observed in each experiment. Lane P, protein only; lanes 0, ssRNA only; lanes 1 to 7 (left panel), ssRNA with twofold dilutions of protein from a final concentration of 128 μM to 2 μM for ssRNA1; lanes 2, 4, 6, and 8 (right panel), ssRNA with fourfold dilutions of protein from 64 μM to 1 μM for ssRNA2. Electrophoretic mobilities of free (f) and bound (b) forms of each ssRNA species are indicated with arrowheads. (c) ssRNA1-binding at variable concentrations of nsp3a(1-112), as calculated from the EMSA data shown in panel b.
Article Snippet: RNase-free DNase I (NEB), T7 endonuclease I (NEB), RNase If (NEB), RNase A (Invitrogen), and RNase T1 (Ambion) cleavage assays were thus performed at 37°C for 1 h with the manufacturer's recommended buffer conditions.
Techniques: Purification, Staining, Electrophoresis, RNA Binding Assay, Incubation, Binding Assay, Concentration Assay