Clarke, P. A., Mathews, M. B. (March 1995) Interactions between the double-stranded RNA binding motif and RNA: definition of the binding site for the interferon-induced protein kinase DAI (PKR) on adenovirus VA RNA. Rna, 1 (1). pp. 7-20. ISSN 1355-8382 (Print)
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Abstract
The protein kinase DAI, the double-stranded RNA activated inhibitor of translation (also known as PKR), regulates cell growth, virus infection, and other processes. DAI represents a class of proteins containing a recently recognized RNA binding motif, the dsRBM, but little is known about the contacts between these proteins and their RNA ligands. In adenovirus-infected cells, DAI activation is prevented by VA RNAI, a highly structured RNA that binds to the kinase. VA RNA contains three chief structural features: a terminal stem, an apical stem-loop, and a complex central domain. We used enzymatic and chemical footprinting to identify the interactions between DAI and VA RNAI. DAI protects the proximal part of the apical stem structure, an adjacent region in the central domain, and a region surrounding a conserved stem in the central domain from nuclease attack. During binding the RNA undergoes a conformational change that is mainly restricted to the central domain. A similar change is induced by magnesium ions alone. Footprinting and interference binding assays using base-specific chemical probes suggest that the protein does not make major contacts with RNA bases. On the other hand, footprinting with probes specific for the RNA backbone shows that DAI engages in a strong interaction with the minor groove of the apical stem and a weaker interaction in the central domain. A truncated form of DAI, p20, containing only the RNA binding domain, gives a similar protection pattern in the apical stem but protects the central domain less effectively. We conclude that the RNA binding domain of DAI interacts directly with the apical stem and central domain of VA RNA, and that other regions of the protein contribute to interactions with the central domain.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | Adenoviruses, Human Base Sequence Binding Sites Enzyme Induction Hydroxyl Radical/pharmacology Interferons/pharmacology Magnesium/pharmacology Molecular Sequence Data Nucleic Acid Conformation/drug effects Peptide Fragments/metabolism Protein Binding Protein-Serine-Threonine Kinases/ metabolism RNA, Double-Stranded/drug effects/ metabolism RNA, Viral/drug effects/ metabolism RNA-Binding Proteins/ metabolism Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. Ribonucleases/pharmacology Thionucleotides eIF-2 Kinase |
| Subjects: | bioinformatics > genomics and proteomics > design > nucleic acid design organism description > virus > adenovirus organism description > animal > mammal > primates > hominids > human bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > RNA binding protein |
| CSHL Authors: | |
| Communities: | CSHL labs |
| Depositing User: | Jessica Koos |
| Date: | March 1995 |
| Date Deposited: | 15 Aug 2014 19:08 |
| Last Modified: | 15 Aug 2014 19:08 |
| PMCID: | PMC1369062 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/30578 |
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