Date of Completion

Summer 6-13-2014

Thesis Advisor(s)

James L. Cole

Honors Major

Molecular and Cell Biology


Biochemistry | Biophysics | Molecular Biology | Structural Biology


Protein Kinase R (PKR) is a key component of the innate immune antiviral response. PKR is activated upon binding to dsRNA. However, recent studies have shown that PKR can also bind to and become activated by duplex RNAs containing complex secondary structure. The mechanism of PKR binding and activation by these RNAs is currently not known. The approach taken here to determine the mechanism of PKR binding by these RNAs is through the development of PKR constructs that are capable of covalently binding to RNAs. Constructs were created by site-specific incorporation of an unnatural, photoactivatable amino acid within PKR. These constructs were incubated with RNA and crosslinked at various UV light wavelengths. Subsequently, SDS-PAGE was performed to detect crosslinking products. The incentive for developing these crosslinking constructs is to use them to map PKR binding on RNAs and to determine the correlation between RNA structural features and PKR activation or inhibition.