Binding of the protein kinase PKR to RNAs with secondary structure defects: role of the tandem A-G mismatch and noncontiguous helixes.
The human interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an antiviral agent that is activated by long stretches of dsRNA. PKR can also be activated or repressed by a series of cellular and viral RNAs containing non-Watson-Crick motifs. PKR has a dsRNA-binding domain (dsRBD) that contains two tandem copies of the dsRNA-binding motif (dsRBM). In vitro selection experiments were carried out to search for RNAs capable of binding to a truncated version of PKR containing the dsRBD. RNA ligands were selected by binding to His6-tagged proteins and chromatography on nickel(II) nitrilotriacetic acid agarose. A series of RNAs was selected that bind either similar to or tighter than a model dsRNA stem loop. Examination of these RNAs by a variety of methods, including sequence comparison, free-energy minimization, structure mapping, boundary experiments, site-directed mutagenesis, and footprinting, revealed protein-binding sites composed of noncontiguous helices. In addition, selected RNAs contained tandem A-G mismatches (5'AG3'/3'GA5'), yet bound to the truncated protein with affinities similar to duplexes containing only Watson-Crick base pairs. The NMR structure of the tandem A-G mismatch in an RNA helix (rGGCAGGCC)2 reveals a global A-form helix with minor perturbations at the mismatch [Wu, M., SantaLucia, J., Jr., and Turner, D. H. (1997) Biochemistry 36, 4449-4460]. This supports the notion that dsRBM-containing proteins can bind to RNAs with secondary structure defects as long as the RNA has an overall A-form geometry. In addition, selected RNAs are able to activate or repress wild-type PKR autophosphorylation as well as its phosphorylation of protein synthesis initiation factor eIF-2, suggesting full-length PKR can bind to and be regulated by RNAs containing a tandem A-G mismatch.
Bevilacqua
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Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309-0215, USA.
George
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Samuel
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Cech
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D001483:Base Sequence; D002846:Chromatography, Affinity; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017433:Protein Structure, Secondary; D012313:RNA / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism; D016415:Sequence Alignment; D019892:eIF-2 Kinase / Q000378:metabolism*
D012330:RNA, Double-Stranded; D012313:RNA; D019892:eIF-2 Kinase