Identification of binding sites for both dsRBMs of PKR on kinase-activating and kinase-inhibiting RNA ligands

Richard J. Spanggord, Momchilo Vuyisich, Peter A. Beal

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates and inhibits the function of the translation initiation factor eIF-2. PKR has a double-stranded RNA-binding domain (dsRBD) composed of two copies of the dsRNA binding motif (dsRBM). PKR's dsRBD is involved in the regulation of the enzyme as dsRNAs of cellular and viral origins bind to the dsRBD, leading to either activation or inhibition of PKR's kinase activity. In this study, we site-specifically modified each of the dsRBMs of PKR's dsRBD with the hydroxyl radical generator EDTA·Fe and performed cleavage studies on kinase-activating and kinase-inhibiting RNAs. These experiments led to the identification of binding sites for the individual dsRBMs on various RNA ligands including a viral activating RNA (TAR from HIV-1), a viral inhibiting RNA (VAI RNA from adenovirus), an aptamer RNA that activates PKR, and a small synthetic inhibiting RNA. These results indicate that some RNAs interact only with one dsRBM, while others can bind both dsRBMs of PKR. In addition, EDTA·Fe modification coupled with site-directed mutagenesis was used to assess the extent of cooperativity in the binding of the two dsRBMs. These experiments support the hypothesis that simultaneous binding of both dsRBMs of PKR occurs on kinase activating RNA ligands.

Original languageEnglish (US)
Pages (from-to)4511-4520
Number of pages10
JournalBiochemistry
Volume41
Issue number14
DOIs
StatePublished - Apr 9 2002
Externally publishedYes

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Phosphotransferases
Binding Sites
RNA
Ligands
Double-Stranded RNA
eIF-2 Kinase
Viral RNA
Prokaryotic Initiation Factor-2
Nucleotide Aptamers
Peptide Initiation Factors
Mutagenesis
Enzymes
Site-Directed Mutagenesis
Hydroxyl Radical
Interferons
HIV-1
Experiments
Chemical activation
Double-Stranded RNA Binding Motif

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of binding sites for both dsRBMs of PKR on kinase-activating and kinase-inhibiting RNA ligands. / Spanggord, Richard J.; Vuyisich, Momchilo; Beal, Peter A.

In: Biochemistry, Vol. 41, No. 14, 09.04.2002, p. 4511-4520.

Research output: Contribution to journalArticle

Spanggord, Richard J. ; Vuyisich, Momchilo ; Beal, Peter A. / Identification of binding sites for both dsRBMs of PKR on kinase-activating and kinase-inhibiting RNA ligands. In: Biochemistry. 2002 ; Vol. 41, No. 14. pp. 4511-4520.
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