Analysis of high affinity binding of PKR to dsRNA

Protein kinase R (PKR) is an interferon-induced kinase which plays a pivotal role in the innate immunity response to viral infection. PKR is activated upon binding to dsRNA. Our previous analysis of PKR binding to dsRNAs ranging from 20-40 bp supports a dimerization model for activation where 30 bp represents the minimal length required to bind two PKR monomers and activate PKR via autophosphorylation. These studies were complicated by the formation of protein-RNA aggregates, particularly at low salt concentrations using longer dsRNAs. Here, we have taken advantage of the enhanced sensitivity afforded using fluorescence-detected analytical ultracentrifugation to reduce the RNA concentrations from micromolar to nanomolar. Under these conditions, we are able to characterize high affinity binding of PKR to longer dsRNAs in 75 mM NaCl. The PKR binding stoichiometries are increased at lower salt but remain lower than those previously obtained for the dsRNA binding domain. The dependence of the limiting PKR binding stoichiometries on dsRNA length does not conform to standard models for nonspecific binding and suggests that binding to longer sequences occurs via a different binding mode with a larger site size. Although dimerization plays a key role in the PKR activation mechanism, the ability o