Date of Completion

12-3-2015

Embargo Period

11-17-2016

Keywords

Protein Kinase R, Dimerization, FRET, Analytical Ultracentrifugation, Unnatural Amino Acid

Major Advisor

Dr. James L. Cole

Associate Advisor

Dr. Nathan N. Alder

Associate Advisor

Dr. Carolyn M. Teschke

Field of Study

Biochemistry

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Protein kinase R (PKR) is a key component of the interferon-induced immune response to viral stress. It contains two N-terminal dsRNA binding domains (dsRBD) and a C-terminal serine/threonine kinase domain separated by a 90 residue unstructured linker of unknown function. PKR is activated by viral dsRNAs via dimerization and autophosphorylation of specific serine and threonine residues. We have developed an assay to measure dimerization of PKR while bound to various dsRNAs using Fӧrster resonance energy transfer. This is the first direct measurement of PKR dimerization upon dsRNAs binding. Our results show that inactive dimers of PKR exist in solution, and that a specific back-to-back dimer configuration is essential for activation. The role of the interdomain linker was determined by characterizing linker deleted constructs of PKR. We propose that the unstructured linker may exist to negatively regulate PKR activation by limiting it’s binding stoichiometry and curbing dsRNA-independent activation. The role of the second dsRNA binding domain was probed by mutating residues likely to contact dsRNA. Collectively, the studies presented here provide new insight on the elusive mechanism of dsRNA induced PKR activation.

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