Specific recognition of HIV TAR RNA by the dsRNA binding domains (dsRBD1-dsRBD2) of PKR.
PKR (double-stranded RNA-dependent protein kinase) is an important component of host defense to virus infection. Binding of dsRNA to two dsRBDs (double-stranded RNA binding domains) of PKR modulates its own kinase activation. How structural features of natural target RNAs, such as bulges and loops, have an effect on the binding to two dsRBDs of PKR still remains unclear. By using ITC and NMR, we show here that both the bulge and loop of TAR RNA are necessary for the high affinity binding to dsRBD1-dsRBD2 of PKR with 1:1 stoichiometry. The binding site for the dsRBD1-dsRBD2 spans from upper bulge to lower stem of the TAR RNA, based on chemical shift mapping. The backbone resonances in the 40 kDa TAR.dsRBD1-dsRBD2 were assigned. NMR chemical shift perturbation data suggest that the beta1-beta2 loop of the dsRBD1 interacts with the TAR RNA, whereas that of the dsRBD2 is less involved in the TAR RNA recognition. In addition, the residues of the interdomain linker between the dsRBD1 and the dsRBD2 also show large chemical perturbations indicating that the linker is involved in the recognition of TAR RNA. The results presented here provide the biophysical and spectroscopic basis for high-resolution structural studies, and show how local RNA structural features modulate recognition by dsRBDs.
Kim
Insil
I
Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126, USA.
Liu
Corey W
CW
Puglisi
Joseph D
JD
D000595:Amino Acid Sequence; D001483:Base Sequence; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*; D000099253:Trans-Activation Responsive RNA-Binding Protein
D012330:RNA, Double-Stranded; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D019892:eIF-2 Kinase; D000099253:Trans-Activation Responsive RNA-Binding Protein