title: Minor-groove recognition of double-stranded RNA by the double-stranded RNA-binding domain from the RNA-activated protein kinase PKR.
abstract: The human double-stranded RNA- (dsRNA) activated protein kinase (PKR) has a dsRNA-binding domain (dsRBD) that contains two tandem copies of the dsRNA-binding motif (dsRBM). The minimal-length polypeptide required to bind dsRNA contains both dsRBMs, as determined by mobility-shift and filter-binding assays. Mobility-shift experiments indicate binding requires a minimum of 16 base pairs of dsRNA, while a minimal-length site for saturation of longer RNAs is 11 base pairs. Bulge defects in the helix disfavor binding, and single-stranded tails do not strongly influence the dsRNA length requirement. These polypeptides do not bind an RNA-DNA hybrid duplex or dsDNA as judged by either mobility-shift or competition experiments, suggesting 2'-OH contacts on both strands of the duplex stabilize binding. Related experiments on chimeric duplexes in which specific sets of 2'-OHs are substituted with 2'-H or 2'-OCH3 reveal that the 2'-OHs required for binding are located along the entire 11 basepair site. These results are supported by Fe(II) EDTA footprinting experiments that show protein-dependent protection of the minor groove of dsRNA. The dependence of dsRNA-protein binding on salt concentration suggests that only one ionic contact is made between the protein and dsRNA phosphate backbone and that at physiological salt concentrations 90% of the free energy of binding is nonelectrostatic. Thus, the specificity of PKR for dsRNA over RNA-DNA hybrids and dsDNA is largely due to molecular recognition of a network of 2'-OHs involving both strands of dsRNA and present along the entire 11 base-pair site.
authors: Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder 80309-0215, USA.
authors: P C
authors: PC
authors: Bevilacqua
authors: T R
authors: TR
authors: Cech
mesh_terms: D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli; D005296:Ferrous Compounds / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D019892:eIF-2 Kinase
chemical_list: D005296:Ferrous Compounds; D009838:Oligodeoxyribonucleotides; D012330:RNA, Double-Stranded; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C055526:Fe(II)-EDTA; D004492:Edetic Acid; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase