Characterization of the solution complex between the interferon-induced, double-stranded RNA-activated protein kinase and HIV-I trans-activating region RNA

Bruce W. Carpick, Vito Graziano, Dieter Schneider, Ratan K. Maitra, Xavier Lee, Bryan R.G. Williams

Research output: Contribution to journalArticleResearchpeer-review

104 Citations (Scopus)

Abstract

The antiviral activity of the interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase (PKR) is mediated through dsRNA binding leading to PKR autophosphorylation and subsequent inhibition of protein synthesis. Previous biochemical studies have suggested that autophosphorylation of PKR occurs via a protein-protein interaction and that PKR can form dimers in vitro. Using four independent biophysical and biochemical methods, we have characterized the solution complex formed between PKR and trans-activating region (TAR) RNA, a 57-nucleotide RNA species with double-stranded secondary structure derived from the human immunodeficieney virus type I genome. Chemical cross-linking and gel filtration analyses of PKR·TAR RNA complexes reveals that TAR RNA addition increases PKR dimerization and results in the formation of a solution complex with a molecular weight of approximately 150,000. Addition of TAR RNA to PKR results in a quenching of tryptophan fluorescence, indicative of a conformational shift. Through small angle neutron scattering analysis, we show that PKR exists in solution predominantly as a dimer, and has an elongated solution structure. Addition of TAR RNA to PKR causes a significant conformational shift in the protein at a 2:1 stoichiometric ratio of protein to RNA. Taken together, these data indicate that the PKR activation complex consists of a protein dimer bound cooperatively to one dsRNA molecule.

Original languageEnglish
Pages (from-to)9510-9516
Number of pages7
JournalThe Journal of Biological Chemistry
Volume272
Issue number14
DOIs
Publication statusPublished - 4 Apr 1997
Externally publishedYes

Cite this