Mechanism of interferon action: RNA-binding activity of full-length and R-domain forms of the RNA-dependent protein kinase PKR--determination of KD values for VAI and TAR RNAs.
The RNA-binding activity of the interferon-inducible, RNA-dependent protein kinase PKR, expressed from the human PKR cDNA, was quantitated using a gel mobility-shift assay. The N-terminal R-domain truncation Wt(1-243) and the full-length catalytic mutant K296R(21-551) were analyzed for their abilities to bind adenovirus VAI RNA, human immunodeficiency virus TAR RNA, and the synthetic homopolymer pI:pC RNA. The N-terminal 243 amino acid residue form of PKR [Wt(1-243)] bound VAI RNA with similar affinity as the 551 amino acid residue full-length catalytic mutant [K296R(1-551)]. The dissociation constant for VAI RNA was approximately 2 x 10(-9) M for both the K296R(1-551) and Wt(1-243) proteins. The K64E mutation significantly impaired the VAI 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 pI: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 x 10(-7) M. These results suggest that the RNA-binding affinity of PKR is approximately 100-fold lower for TAR RNA than for either VAI RNA or pI:pC RNA and that the full-length and N-terminal R-domain forms of PKR bind RNA with similar affinity.
McCormack
S J
SJ
Department of Biological Sciences, University of California, Santa Barbara 93106.
Samuel
C E
CE
D000256:Adenoviridae / Q000235:genetics; D006678:HIV / Q000235:genetics; D016325:HIV Long Terminal Repeat / Q000235:genetics; D007372:Interferons / Q000494:pharmacology*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015347:RNA Probes; D012367:RNA, Viral / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D019892:eIF-2 Kinase
D015347:RNA Probes; D012367:RNA, Viral; D011994:Recombinant Proteins; D007372:Interferons; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase