X-ray structure and activities of an essential Mononegavirales L-protein domain

Paesen, Guido C.; Collet, Axelle; Sallamand, Corinne; Debart, Françoise; Vasseur, Jean-Jacques; Canard, Bruno; Decroly, Etienne; Grimes, Jonathan M.

Characterization of RNA-CR-VI þ interactions. Using T4 RNA ligase 1 (20 units; New England Biolabs), [5 0 -32 P]-pCp (1.1 mCi) was ligated to the 3 0 -ends of the RNA substrates (10 mM) in 50 mM Tris-HCl (pH 7.8), 10 mM MgCl 2 , 10 mM DTT and 1 mM ATP (16°C, overnight). Ligase was removed by RNA precipitation in 3 M sodium acetate supplemented with 1 mg ml À 1 of glycogen (Thermo Scientific). The radiolabelled RNA was incubated (15 min, 37°C) with increasing concentrations of CR-VI þ , in 20 mM Tris-HCl (pH 8.5), 1 mM DTT, 10% glycerol and 30 mM NaCl. The reaction mixtures were spotted onto nitrocellulose (GE Healthcare) using a manifold-1 dot-blot apparatus (Whatman) and washed with 20 mM Tris-HCl (pH 8.5), 1 mM DTT and 50 mM NaCl. Membrane-bound RNA was quantified by phosphor-imaging. Dissociation constants (K D s) were determined using Hill slope curve fitting (Prism).