Straightening of bulged RNA by the double-stranded RNA-binding domain from the protein kinase PKR.
The human interferon-induced protein kinase, PKR, is an antiviral agent that is activated by long stretches of double-stranded (ds)RNA. PKR has an N-terminal dsRNA-binding domain that contains two tandem copies of the dsRNA-binding motif and interacts with dsRNA in a nonsequence-specific fashion. Surprisingly, PKR can be regulated by certain viral and cellular RNAs containing non-Watson-Crick features. We found that RNAs containing bulges in the middle of a helix can bind to p20, a C-terminal truncated PKR containing the dsRNA-binding domain. Bulges are known to change the global geometry of RNA by bending the helical axis; therefore, we investigated the conformational changes of bulged RNA caused by PKR binding. A 66-mer DNA-RNA(+/- A(3) bulge)-DNA chimera was constructed and annealed to a complementary RNA strand. This duplex forces the protein to bind in the middle. A 66-mer duplex with a top strand composed of DNA-DNA(+/-A(3) bulge)-RNA was used as a control. Gel mobility-shift changes among the RNA-protein complexes are consistent with straightening of bulged RNA on protein binding. In addition, a van't Hoff analysis of p20 binding to bulged RNA reveals a favorable DeltaDeltaH degrees and an unfavorable DeltaDeltaS degrees relative to binding to straight dsRNA. These thermodynamic parameters are in good agreement with predictions from a nearest-neighbor analysis for RNA straightening and support a model in which the helical junction flanking the bulge stacks on protein binding. The ability of dsRNA-binding motif proteins to recognize and straighten bent RNA has implications for modulating the topology of RNAs in vivo.
Zheng
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Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA.
Bevilacqua
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PC
D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics; D019892:eIF-2 Kinase / Q000378:metabolism*
D009692:Nucleic Acid Heteroduplexes; D012330:RNA, Double-Stranded; D019892:eIF-2 Kinase