Binding of the protein kinase PKR to RNAs with secondary structure defects: Role of the tandem A - G mismatch and noncontigous helixes

Philip C. Bevilacqua, Cyril X. George, Charles E. Samuel, Thomas R. Cech

Research output: Contribution to journalArticlepeer-review

93 Scopus citations

Abstract

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 ((3'GA5')/(5'AG3')) 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.

Original languageEnglish (US)
Pages (from-to)6303-6316
Number of pages14
JournalBiochemistry
Volume37
Issue number18
DOIs
StatePublished - May 5 1998

All Science Journal Classification (ASJC) codes

  • Biochemistry

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