INTERACTIONS BETWEEN THE DOUBLE-STRANDED-RNA BINDING MOTIF AND RNA - DEFINITION OF THE BINDING-SITE FOR THE INTERFERON-INDUCED PROTEIN-KINASE DAI (PKR) ON ADENOVIRUS VA RNA

The protein kinase DAI, the double-stranded RNA activated inhibitor of translation (also known as PKR), regulates cell growth, virus infecti on, and other processes. DAI represents a class of proteins containing a recently recognized RNA binding motif, the dsRBM, but little is kno wn about the contacts between these proteins and their RNA ligands. In adenovirus-infected cells, DAI activation is prevented by VA RNA(I), a highly structured RNA that binds to the kinase. VA RNA contains thre e chief structural features: a terminal stem, an apical stem-loop, and a complex central domain. We used enzymatic and chemical footprinting to identify the interactions between DAI and VA RNA(I). DAI protects the proximal part of the apical stem structure, an adjacent region in the central domain, and a region surrounding a conserved stem in the c entral domain from nuclease attack. During binding the RNA undergoes a conformational change that is mainly restricted to the central domain . A similar change is induced by magnesium ions alone. Footprinting an d interference binding assays using base-specific chemical probes sugg est that the protein does not make major contacts with RNA bases. On t he other hand, footprinting with probes specific for the RNA backbone shows that DAI engages in a strong interaction with the minor groove o f the apical stem and a weaker interaction in the central domain. A tr uncated form of DAI, p20, containing only the RNA binding domain, give s a similar protection pattern in the apical stem but protects the cen tral domain less effectively. We conclude that the RNA binding domain of DAI interacts directly with the apical stem and central domain of V A RNA, and that other regions of the protein contribute to interaction s with the central domain.