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.

By analysing the steady-state ribonucleotide-protein (RNP) complexes formed through binding of nsp8 to 5 0 32 P-labelled dsRNA ( Figure 3A) , we estimated the nsp8 dissociation constant (K d ) for dsRNA to be $3.3 mM ( Figure 3F) , which is about $25-fold higher than the apparent K d of nsp12 under comparable conditions (10) . A comprehensive analysis of the influence of nsp7 on nsp8-dependent RNA binding required an nsp8 mutant that was incapable of RNA binding. To this end, we engineered an alanine substitution of the conserved residue K58, which resides in nsp8's proposed dsRNA-binding channel [residues 55-78 (13)]. As is evident from the electromobility shift assay in Figure 3B , this mutation was sufficient to significantly disrupt RNA binding. As a control, we also performed an aspartate-to-alanine substitution at position 52, which is partially conserved, yet not expected to participate in RNA backbone binding due to its negative charge and position just outside the proposed RNA binding channel. Indeed, the D52A mutation only induced a migratory shift of the dominant RNP signal towards the anode, likely as a result of the lost negative charge ( Figure 3C ).