The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension

te Velthuis, Aartjan J.W.; van den Worm, Sjoerd H. E.; Snijder, Eric J.

A dilution series of 0-5 mM SARS-CoV nsp8 in storage buffer was incubated for 10 min at 20 C with 0.2 nM of 32 P-labelled duplex RNA. Subsequently, samples were directly loaded onto 8% polyacrylamide gels containing 5% glycerol and 0.5x TGE (25 mM Tris, 190 mM glycine and 10 mM EDTA) buffer and run at 150 V for 1 h at 4 C. Gels were dried on Whatman filter paper and bands were quantified by phosphorimaging using a Typhoon variable mode scanner (GE Healthcare) and ImageQuant TL 7.0 software (GE Healthcare) as described elsewhere (10) . Using the Matlab 2009a Curve Fitting Toolbox, the percentage of bound RNA was fit to the Hill equation, which is defined as: RNA bound ¼ b Á ½nsp8 n =ðK n d +½nsp8 n Þ. Here b is the upper binding limit, ½nsp8 the nsp8 concentration, n the Hill coefficient and K d the dissociation constant.