Mechanism of interferon action: RNA-binding activity of full-length and R-domain forms ofthe RNA-dependent protein kinase PKR — Determination of KD values for VAI and TAR RNAs

AbstractThe RNA-binding activity of the interferon-inducible, RNA-dependent protein kinase PKR, expressed from the human PKRcDNA, was quantitated using a gel mobility-shift assay. The N-terminal R-domain truncation Wt(1–243) and the full-length catalytic mutant K296R(1–551) were analyzed for their abilities to bind adenovirus VA, RNA, human immunodeficiency virus TAR RNA, and the synthetic homopolymer pl:pC RNA. The N-terminal 243 amino acid residue form of PKR [Wt(1–243)] bound VA, RNA with similar affinity as the 551 amino acid residue full-length catalytic mutant [K296R(1–551)]. The dissociation constant for VA, RNA was approximately 2 × 10−9 M for both the K296R(1–551) and Wt(1–243) proteins. The K64E mutation significantly impaired the VA, RNA-binding activity as measured with the full-length double-point mutant PKR protein, K64E/K296R(1–551). Using a gel-shift competition assay, the dissociation constants of K296R(1–551) and Wt(1–243) for VAI(1–160) RNA and pl:pC RNA were comparable. By contrast, the dissociation constants of K296R(1–551) and Wt(1–243) for TAR(1–82) RNA were both about 1 × 10−7 M. These results suggest that the RNA-binding affinity of PKR is approximately 100-fold lower for TAR RNA than for either VA, RNA or pl:pC RNA and that the full-length and N-terminal R-domain forms of PKR bind RNA with similar affinity