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.

With the results obtained with these control proteins in mind, we next explored the contribution of nsp7 to RNA binding by the nsp(7+8) complex. We used a fixed concentration of nsp7 and added either wild-type or mutant nsp8 up to the point where the nsp7:nsp8 ratio reached equimolarity. No RNA binding was observed in the absence of nsp8, but upon nsp(7+8) complex formation the amount of bound dsRNA rapidly increased ( Figure 3D ). Indicative of successful complex formation, we also observed a shift in the molecular weight of the major RNP complex formed ( Figure 3D ). Western blot analysis confirmed that both nsp7 and nsp8 were present at this position in the gel (not shown), but due to the generally unpredictable migration behaviour of proteins and RNPs in native PAGE, it was not possible to assess whether this band indeed corresponded to the nsp(7+8) hexadecamer. The K d of the nsp(7+8) complex was estimated at $1.2 mM, about 3-fold lower than that of nsp8 alone ( Figure 3F ).