Date of Completion

5-15-2014

Embargo Period

5-14-2014

Major Advisor

James L. Cole

Associate Advisor

Nathan N. Alder

Associate Advisor

Carolyn M. Teshcke

Field of Study

Structural Biology and Biophysics

Degree

Doctor of Philosophy

Open Access

Campus Access

Abstract

Protein kinase R (PKR) is a component of the innate immune pathway that binds to dsRNA and is activated upon subsequent dimerization and autophosphorylation. Adenovirus virus-associated RNA I (VAI) is a non-coding transcript that binds PKR with high affinity but functions to inhibit the activity of PKR. The secondary structure of VAI consists of three domains: an apical stem loop, a central domain, and a terminal stem. Although VAI has been extensively studied, its three dimensional structure and the mechanism of inhibition of PKR are not known. We show that the interaction between VAI and PKR is dependent on divalent ion. Although two PKR molecules bind to VAI in the absence of magnesium, only one PKR binds in the presence of divalent ion. PKR binds to the double-stranded regions in the apical stem and central domain regions and both of these regions are required to form a specific, high-affinity PKR-binding site. PKR binds the isolated terminal stem with low affinity and deletion of this region from VAI does not affect PKR binding or activation. We use biochemical and biophysical methods to characterize the structure of VAI. Chemical probing and mutagenesis experiments define the secondary structure of VAI and reveal a tertiary contact between two loop regions in the central domain. These data are used to generate a three dimensional structural model of VAI that is supported by small angle x-ray scattering experiments. These studies provide the first picture of how PKR recognizes a highly structured RNA inhibitor.

Download
COinS